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Substantial spine loss in striatal medium spiny neurons (MSNs) and abnormal behaviors are common features of Parkinson's disease (PD). The caudate putamen (CPu) mainly contains MSNs expressing dopamine D1 receptor (dMSNs) and dopamine D2 receptor (iMSNs) exerting critical effects on motor and cognition behavior. However, the molecular mechanisms contributing to spine loss and abnormal behaviors in dMSNs and iMSNs under parkinsonian state remain unknown. In the present study, we revealed that Cell division control protein 42 (Cdc42) signaling was significantly decreased in the caudate putamen (CPu) in parkinsonian mice. In addition, overexpression of constitutively active Cdc42 in the CPu reversed spine abnormalities and improved the behavior deficits in parkinsonian mice. Utilizing conditional dopamine D1 receptor (D1R) or D2 receptor (D2R) knockout mice, we found that such a decrease under parkinsonian state was further reduced by conditional knockout of the D2R but not D1R. Moreover, the thin spine loss in iMSNs and deficits in motor coordination and cognition induced by conditional knockout of D2R were reversed by overexpression of constitutively active Cdc42 in the CPu. Additionally, conditional knockout of Cdc42 from D2R‐positive neurons in the CPu was sufficient to induce spine and behavior deficits similar to those observed in parkinsonian mice. Overall, our results indicate that impaired Cdc42 signaling regulated by D2R plays an important role in spine loss and behavioral deficits in PD. [ABSTRACT FROM AUTHOR]

Curcumin is a polyphenol compound that exhibits multiple physiological activities. To elucidate the mechanisms by which curcumin affects systemic amyloidosis, we investigated amyloid deposition and molecular changes in a mouse model of amyloid apolipoprotein A-II (AApoAII) amyloidosis, in which mice were fed a curcumin-supplemented diet. Curcumin supplementation for 12 weeks significantly increased AApoAII amyloid deposition relative to controls, especially in the liver and spleen. Liver weights and plasma ApoA-II and high-density lipoprotein concentrations were significantly elevated in curcumin-supplemented groups. RNA-sequence analysis revealed that curcumin intake affected hepatic lipid metabolism via the peroxisome proliferator-activated receptor (PPAR) pathway, especially PPARa activation, resulting in increased Apoa2 mRNA expression. The increase in liver weights was due to activation of PPARa and peroxisome proliferation. Taken together, these results demonstrate that curcumin is a PPARa activator and may affect expression levels of proteins involved in amyloid deposition to influence amyloidosis and metabolism in a complex manner. [ABSTRACT FROM AUTHOR]

Intraocular pressure (IOP) is the primary risk factor for developing glaucoma, yet little is known about the contribution of genomic background to IOP regulation. The present study leverages an array of systems genetics tools to study genomic factors modulating normal IOP in the mouse. The BXD recombinant inbred (RI) strain set was used to identify genomic loci modulating IOP. We measured the IOP in a total of 506 eyes from 38 different strains. Strain averages were subjected to conventional quantitative trait analysis by means of composite interval mapping. Candidate genes were defined, and immunohistochemistry and quantitative PCR (qPCR) were used for validation. Of the 38 BXD strains examined the mean IOP ranged from a low of 13.2mmHg to a high of 17.1mmHg. The means for each strain were used to calculate a genome wide interval map. One significant quantitative trait locus (QTL) was found on Chr.8 (96 to 103 Mb). Within this 7 Mb region only 4 annotated genes were found: Gm15679, Cdh8, Cdh11 and Gm8730. Only two genes (Cdh8 and Cdh11) were candidates for modulating IOP based on the presence of non-synonymous SNPs. Further examination using SIFT (Sorting Intolerant From Tolerant) analysis revealed that the SNPs in Cdh8 (Cadherin 8) were predicted to not change protein function; while the SNPs in Cdh11 (Cadherin 11) would not be tolerated, affecting protein function. Furthermore, immunohistochemistry demonstrated that CDH11 is expressed in the trabecular meshwork of the mouse. We have examined the genomic regulation of IOP in the BXD RI strain set and found one significant QTL on Chr. 8. Within this QTL, there is one good candidate gene, Cdh11. [ABSTRACT FROM AUTHOR]

Changes in chloride reversal potential in rat spinal cord neurons have previously been associated with persistent pain in nerve injury and inflammation models. These changes correlate with a decrease in the expression of the potassium chloride transporter, KCC2, and with increases in neuronal excitability. Here, we test the hypothesis that similar changes occur in mice with neuropathic pain induced by chronic constriction injury of the trigeminal infraorbital nerve (CCI-ION). This model allows us to distinguish an acute pain phase (3-5 days after injury) from a persistent pain phase (12-14 days after CCI-ION). Chronic constriction injury of the trigeminal infraorbital nerve induced significant decreases in mechanical pain thresholds in both the acute and persistent phases. To estimate GABAA reversal potentials in neurons from trigeminal nucleus caudalis, we obtained perforated patch recordings in vitro. GABAA reversal potential decreased by 8% during the acute phase in unidentified neurons, but not in GABAergic interneurons. However, at 12 to 14 days after CCI-ION, GABAA reversal potential recovered to normal values. Quantitative real-time polymerase chain reaction analysis revealed no significant changes, at either 3 to 5 days or 12 to 14 days after CCI-ION, in either KCC2 or NKCC1. These findings suggest that CCI-ION in mice results in transient and modest changes in chloride reversal potentials, and that these changes may not persist during the late phase. This suggests that, in the mouse model of CCI-ION, chloride dysregulation may not have a prominent role in the central mechanisms leading to the maintenance of chronic pain. [ABSTRACT FROM AUTHOR]

Aims: This study observes the effects of phlorizin on diabetic nephrology in db/db diabetic miceand explores possible underlying mechanisms. Methods: Sixteen diabetic db/db mice and eight age-matched db/m mice were divided into three groups: vehicle-treated diabetic group (DM group), diabetic group treated with phlorizin (DMT group) and normal control group (CC group). Phlorizin was given in normal saline solution by intragastric administration for 10 weeks. Differentially expressed proteins in three groups were identified using iTRAQ quantitative proteomics and the data were further analyzed with ingenuity pathway analysis. Results: The body weight and serum concentrations of fasting blood glucose (FBG), advanced glycation end products (AGEs), total cholesterol, triglycerides, blood urea nitrogen, creatinine and 24-h urine albumin were increased in the DM group compared to those of the CC group (P < 0.05), and they were decreased by treatment with phlorizin (P < 0.05). Morphologic observations showed phlorizin markedly attenuated renal injury. Phlorizin prevented diabetic nephropathy by regulating the expression of a series of proteins involved in renal and urological disease, molecular transport, free radical scavenging, and lipid metabolism. Conclusions: Phlorizin protects mice from diabetic nephrology and thus may be a novel therapeutic approach for the treatment of diabetic nephrology. [ABSTRACT FROM AUTHOR]

Highlights: [•] Running wheel (RW) counteracted the cognitive deficits induced by TBI. [•] RW restored the neuron and synaptic density loss associated with TBI. [•] RW increased the levels of COX I, II, III, BDNF, synapsin I and SYP. [•] RW switched the mitochondrial CcO activity and ATP amounts. [•] COX might play an important role in exercise's cognitive effects in TBI. [ABSTRACT FROM AUTHOR]

Background: Toll-like receptors (TLRs), as major innate immune mediators, may be involved in clearance of cerebral amyloid-β (Aβ) deposits. Recently, a novel TLR9 signaling pathway has been uncovered, which is functionally associated with the immune inflammatory response and reducing Aβ burden in Alzheimer's disease (AD) mice. Therefore, TLR9 might represent a reasonable functional candidate gene for AD. Findings: Our study investigated 1,133 sporadic late-onset AD (LOAD) and 1,159 healthy controls matched for sex and age in a large Han Chinese population. One selected functional rs187084 polymorphism within the TLR9 gene was genotyped by polymerase chain reaction-ligase detection reaction in a case–control associated study. The TLR9 rs187084 variant homozygote GG was significantly associated with a decreased LOAD risk after adjusting for age, gender, and ApoE ε4 status by logistic regression analysis (P = 0.035). Our result showed significant evidence of the interaction of ApoE ε4 with rs187084. When we further stratified our data by the ApoE ε4 status, we detected significant differences in the genotype and allele distributions of rs187084 between LOAD patients and controls in ApoE ε4 carriers (P < 0.001, P = 0.003, respectively). Moreover, we examined TLR9 expression in peripheral blood monocytes by flow cytometry, and the GG genotype of the TLR9 rs187084 polymorphism was associated with a higher TLR9 expression than two other genotypes in LOAD patients. Conclusion: Our findings support the hypothesis that the TLR9 polymorphism may modify LOAD risk in the Han Chinese population. [ABSTRACT FROM AUTHOR]

Background: Bladder transitional cell carcinoma greatly threatens human health all over the world. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows a strong apoptosis-inducing effect on a variety of cancer cells including bladder cancer. However, adenovirus-mediated TRAIL expression still showed cytotoxicity to normal cells mainly due to lack of tumor specificity. Methods: To solve the problem, we applied miRNA response elements (MREs) of miR-1, miR-133 and miR-218 to confer TRAIL expression with specificity to bladder cancer cells. Results: Expression of miR-1, miR-133 and miR-218 was greatly decreased in bladder cancer than normal bladder tissue. Luciferase assay showed that application of the 3 MREs was able to restrain exogenous gene expression to within bladder cancer cells. Subsequently, we constructed a recombinant adenovirus with TRAIL expression regulated by MREs of miR-1, miR-133 and miR-218, namely Ad-TRAIL-MRE-1-133-218. qPCR, immunoblotting and ELISA assays demonstrated that Ad-TRAIL-MRE-1-133-218 expressed in bladder cancer cells, rather than normal bladder cells. The differential TRAIL expression also led to selective apoptosis-inducing and growth-inhibiting effect of Ad-TRAIL-MRE-1-133-218 on bladder cancers. Finally, bladder cancer xenograft in mouse models further confirmed that Ad-TRAIL-MRE-1-133-218 effectively suppressed the growth of bladder cancers. Conclusions: Collectively, we demonstrated that MREs-based TRAIL delivery into bladder cancer cells was feasible and efficient for cancer gene therapy. [ABSTRACT FROM AUTHOR]

The mouse incisor is a valuable but under-utilized model organ for studying the behavior of adult stem cells. This remarkable tooth grows continuously throughout the animal's lifetime and houses two distinct epithelial stem cell niches called the labial and lingual cervical loop (laCL and liCL, respectively). These stem cells produce progeny that undergo a series of well-defined differentiation events en route to becoming enamel-producing ameloblasts. During this differentiation process, the progeny move out of the stem cell niche and migrate toward the distal tip of the tooth. Although the molecular pathways involved in tooth development are well documented, little is known about the roles of miRNAs in this process. We used microarray technology to compare the expression of miRNAs in three regions of the adult mouse incisor: the laCL, liCL, and ameloblasts. We identified 26 and 35 differentially expressed miRNAs from laCL/liCL and laCL/ameloblast comparisons, respectively. Out of 10 miRNAs selected for validation by qPCR, all transcripts were confirmed to be differentially expressed. In situ hybridization and target prediction analyses further supported the reliability of our microarray results. These studies point to miRNAs that likely play a role in the renewal and differentiation of adult stem cells during stem cell-fueled incisor growth. [ABSTRACT FROM AUTHOR]

Certain murine leukemia viruses (MLVs) can induce progressive noninflammatory spongiform neurodegeneration similar to that caused by prions. The primary MLV determinants responsible have been mapped to within the env gene; however, it has remained unclear how env mediates disease, whether non-Env viral components are required, and what central nervous system (CNS) cells constitute the critical CNS targets. To address these questions, we examined the effect of transplanting engraftable C17.2 neural stem cells engineered to pseudotype, disseminate, and trans-complement neurovirulent (CasBrE, CasE, and CasES) or non-neurovirulent (Friend and SFF-FE) env sequences (SU or SU/TM) within the CNS using either the "non-neurovirulent" amphotropic helper virus, 4070A, or pgag-polgpt (a nonpackaged vector encoding Gag-Pol). These studies revealed that acute MLV-induced spongiosis results from two separable activities of Env. First, Env causes neuropathology through unique viral targeting within the CNS, which was efficiently mediated by ecotropic Envs (CasBrE and Friend), but not 4070A amphotropic Env. Second, Env induces spongiosis through a toxin activity that is MLV-receptor independent and does not require the coexpression of other viral structural proteins. CasBrE and 4070A Envs possess the toxin activity, whereas Friend Env does not. Although the identity of the critical viral target cell(s) remains unresolved, our results appear to exclude microglia and oligodendrocyte lineage cells, while implicating viral entry into susceptible neurons. Thus, MLV-induced disease parallels prionopathies in that a single protein, Env, mediates both the CNS targeting and the toxicity of the infectious agent that manifests itself as progressive vacuolar neurodegeneration. [ABSTRACT FROM AUTHOR]

Integration of muscle, connective tissue and skeletal patterning during development is essential for proper functioning of the musculoskeletal system. How this integration is achieved is poorly understood. There is ample evidence suggesting that skeletal pattern is programmed autonomously, whereas muscle pattern is, for the most part, programmed non-cell-autonomously. Connective tissues depend upon both muscle and skeletal tissues for their proper survival and development. Here, we employed a novel approach to dissect the coordination of musculoskeletal patterning during mouse limb development. Using both conditional gain- and loss-of-function approaches, we selectively deleted or activated the LIM-homeodomain transcription factor Lmx1b in skeletal progenitors using a Sox9-Cre knock-in allele. As Lmx1b is both necessary and sufficient to specify dorsal pattern, this approach allowed us to investigate the effect of selectively deleting or activating Lmx1b in skeletal progenitors on muscle, connective and skeletal tissues during limb development. Our results indicate that whereas Lmx1b activity is required autonomously in skeletal progenitors to direct dorsal pattern, loss or gain of Lmx1b activity in skeletal progenitors has no effect on muscle or connective tissue patterning. Hence, we show for the first time that skeletal and connective tissue patterning can be uncoupled, indicating a degree of autonomy in the formation of the musculoskeletal system. [ABSTRACT FROM AUTHOR]

: Aims Lipopolysaccharide (LPS) induces cardiomyocyte caspase-3 activation and proinflammatory factors, in particular tumour necrosis factor-alpha (TNF-α) production, both of which contribute to myocardial dysfunction during sepsis. The present study was to investigate the roles of calpain/calpastatin system in cardiomyocyte caspase-3 activation, TNF-α expression, and myocardial dysfunction during LPS stimulation. : Methods and results In cultured adult rat cardiomyocytes, LPS (1 µg/mL) induced calpain and caspase-3 activity, and up-regulated TNF-α expression. These effects of LPS were abrogated by over-expression of calpastatin, an endogenous calpain inhibitor, transfection of calpain-1 siRNA, or various pharmacological calpain inhibitors. Furthermore, blocking gp91phox-NADPH oxidase prevented calpain and caspase-3 activation and decreased TNF-α expression in LPS-stimulated cardiomyocytes. To investigate the role of calpastatin in endotoxaemia, transgenic mice with calpastatin over-expression (CAST-Tg) and wild-type mice were treated with LPS (4 mg/kg, i.p.) or saline in the presence of calpain inhibitor-III (10 mg/kg, i.p.) for 4 h, and their heart function was measured with a Langendorff system. Over-expression of calpastatin significantly attenuated myocardial dysfunction (P < 0.05). Consistently, calpain activity, caspase-3 activity, and TNF-α expression were also reduced in CAST-Tg and calpain inhibitor-III compared with wild-type and vehicle-treated hearts, respectively. : Conclusion gp91phox-NADPH oxidase-mediated calpain-1 activation induces caspase-3 activation and TNF-α expression in cardiomyocytes during LPS stimulation. Over-expression of calpastatin inhibits calpain activation and improves myocardial function in endotoxaemia. The present study suggests that targeting calpain/calpastatin system may be a potential therapeutic intervention for septic hearts. [ABSTRACT FROM AUTHOR]

The article presents a quantitative proteomics study depicting the protective results of grape seed procyanidin B2 (GSPB2) on diabetic cardiomyopathy in db/db mice, a model of obesity mice. It states the increased in the number of diabetes mellitus incidence worldwide with common complications such as independent of coronary heart disease and atherosclerosis. It also emphasizes the characterization of diabetic cardiomyopathy and its structural and functional changes in the heart.

A series of novel helicid derivatives containing 3,4-dihydropyrimidin-2(1 H)-one and 3,4-dihydropyrimidine2(1 H)-thione moiety ( 3a–3f and 4a–4f) were synthesized starting from helicid. The structure of the new compounds were characterized by 1H NMR, IR and HR-MS spectra. The sedative-hypnotic activities of the target compounds were evaluated using the test of spontaneous locomotor activity in mice. All of the derivatives produced moderate to high activities; in particular, compound 4a presented the most potent sedative-hypnotic effect in comparison to the other derivatives, and derivatives 3a, 3c, 3d, 3e and 3f also showed potent activities. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Hyperglycemia induces reactive oxygen species (ROS) and apoptosis in cardiomyocytes, which contributes to diabetic cardiomyopathy. The present study was to investigate the role of Rac1 in ROS production and cardiomyocyte apoptosis during hyperglycemia. RESEARCH DESIGN AND METHODS--Mice with cardiomyocyte-specific Rac1 knockout (Rac1-ko) were generated. Hyperglycemia was induced in Rac1-ko mice and their wild-type littermates by injection of streptozotocin (STZ). In cultured adult rat cardiomyocytes, apoptosis was induced by high glucose. RESULTS--The results showed a mouse model of STZ-induced diabetes, 7 days of hyperglycemia-upregulated Rac1 and NADPH oxidase activation, elevated ROS production, and induced apoptosis in the heart. These effects of hyperglycemia were significantly decreased in Rac1-ko mice or wild-type mice treated with apocynin. Interestingly, deficiency of Rac1 or apocynin treatment significantly reduced hyperglycemia-induced mitochondrial ROS production in the heart. Deficiency of Rac1 also attenuated myocardial dysfunction after 2 months of STZ injection. In cultured cardiomyocytes, high glucose upregulated Rac1 and NADPH oxidase activity and induced apoptotic cell death, which were blocked by overexpression of a dominant negative mutant of Rac1, knockdown of gp91[sup phox] or p47[sup phox], or NADPH oxidase inhibitor. In type 2 diabetic db/db mice, administration of Rac1 inhibitor, NSC23766, significantly inhibited NADPH oxidase activity and apoptosis and slightly improved myocardial function. CONCLUSIONS--Rac1 is pivotal in hyperglycemia-induced apoptosis in cardiomyocytes. The role of Rac1 is mediated through NADPH oxidase activation and associated with mitochondrial ROS generation. Our study suggests that Rac1 may serve as a potential therapeutic target for cardiac complications of diabetes. Diabetes 58:2386-2395, 2009 [ABSTRACT FROM AUTHOR]

Background: Certain murine leukemia viruses (MLVs) are capable of inducing progressive spongiform motor neuron disease in susceptible mice upon infection of the central nervous system (CNS). The major CNS parenchymal target of these neurovirulent retroviruses (NVs) are the microglia, whose infection is largely coincident with neuropathological changes. Despite this close association, the role of microglial infection in disease induction is still unknown. In this paper, we investigate the interaction of the highly virulent MLV, FrCasE, with microglia ex vivo to evaluate whether infection induces specific changes that could account for neurodegeneration. Specifically, we compared microglia infected with FrCasE, a related non-neurovirulent virus (NN) F43/Fr57E, or mock-infected, both at a basic virological level, and at the level of cellular gene expression using quantitative real time RT-PCR (qRT-PCR) and Afffymetrix 430A mouse gene chips. Results: Basic virological comparison of NN, NV, and mock-infected microglia in culture did not reveal differences in virus expression that provided insight into neuropathogenesis. Therefore, microglial analysis was extended to ER stress gene induction based on previous experiments demonstrating ER stress induction in NV-infected mouse brains and cultured fibroblasts. Analysis of message levels for the ER stress genes BiP (grp78), CHOP (Gadd153), calreticulin, and grp58 in cultured microglia, and BiP and CHOP in microglia enriched fractions from infected mouse brains, indicated that FrCasE infection did not induce these ER stress genes either in vitro or in vivo. To broadly identify physiological changes resulting from NV infection of microglia in vitro, we undertook a gene array screen of more than 14,000 well-characterized murine genes and expressed sequence tags (ESTs). This analysis revealed only a small set of gene expression changes between infected and uninfected cells (<18). Remarkably, gene array comparison of NN- and NVinfected microglia revealed only 3 apparent gene expression differences. Validation experiments for these genes by Taqman real-time RT-PCR indicated that only single Ig IL-1 receptor related protein (SIGIRR) transcript was consistently altered in culture; however, SIGIRR changes were not observed in enriched microglial fractions from infected brains. Conclusion: The results from this study indicate that infection of microglia by the highly neurovirulent virus, FrCasE, does not induce overt physiological changes in this cell type when assessed ex vivo. In particular, NV does not induce microglial ER stress and thus, FrCasE-associated CNS ER stress likely results from NV interactions with another cell type or from neurodegeneration directly. The lack of NV-induced microglial gene expression changes suggests that FrCasE either affects properties unique to microglia in situ, alters the expression of microglial genes not represented in this survey, or affects microglial cellular processes at a post-transcriptional level. Alternatively, NV-infected microglia may simply serve as an unaffected conduit for persistent dissemination of virus to other neural cells where they produce acute neuropathogenic effects. [ABSTRACT FROM AUTHOR]

Insulin resistance induced by a high-fat diet (HFD) is related to metabolic diseases, and sterol O-acyltransferase 1 (SOAT1) is a key enzyme for the biosynthesis of cholesteryl ester. In the present study, wild-type (WT) mice and SOAT1-knockout (KO) mice with a C57BL6 background fed a HFD were used to explore the role of SOAT1 in the hypothalamus. The results show that the WT mice exhibited a significant increase in body weight as well as hepatic histologic changes; they also had a lower glucose and insulin tolerance than the WT mice fed a normal diet. However, the metabolic syndrome was attenuated in the SOAT1-KO HFD-fed mice. With regard to brain function, the SOAT1-KO HFD-fed mice showed improved cognitive function; they also manifested reduced levels of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, which would otherwise be raised by a HFD. In addition, the HFD led to the overexpression of GFAP and phosphorylated NF-κB in the hypothalamus, changes that were reversed in the SOAT1-KO HFD-fed mice. Moreover, SOAT1-KO mice improved HFD-caused defective hypothalamic insulin resistance, as evidenced by the upregulation of p-insulin receptor (INSR), p-AKT and p-glycogen synthase kinase (GSK)-3β, while the downregulation of p-AMP-activated protein kinase (AMPK)–α and p-acetyl-CoA carboxylase (ACC)–α. In addition, similar results were observed in high fructose (HFR)–stimulated astrocytes (ASTs) isolated from WT or KO mice. These results suggest that SOAT1 plays an important role in hypothalamic insulin sensitivity, linked to cognitive impairment, in HFD-fed mice. [ABSTRACT FROM AUTHOR]

• 7-O-(2-(Propylamino)-2-oxoethyl) hesperetin (HD-4d), a new synthesized compound in our laboratory. • HD-4d has obvious anti-inflammatory effect in vivo and in vitro. • HD-4d promoted NLRP12 expression in EtOH and lipopolysaccharide (LPS)-induced RAW264.7 cells and alcohol-induced mouse liver. • HD-4d can suppress p -P65 expression through activating NLRP12 expression. Alcoholic liver disease (ALD) is a liver disease caused by long-term heavy drinking. Alcoholic liver injury is a part of alcoholic liver disease. A large number of studies have shown that alcohol metabolism and endotoxin / lipopolysaccharide (LPS) and cycles can cause massive activation of macrophages, leading alcoholic liver injury. Hesperetin is a dihydro-flavonoid extracted from the fruits of Citrus in Rutaceae. It has a variety of pharmacological activities, including antibacterial, anti-inflammatory, antioxidant and so on, but recent studies have shown that hesperetin derivatives have stronger anti-inflammatory effects than hesperetin. In order to improve the anti-inflammatory activity of hesperetin, our group used ethyl-bromoacetate to replace the hydroxyl group at the 7 position of hesperetin to obtain the hesperetin derivative 7-O-(2-(Propylamino)-2-oxoethyl) hesperetin (HD-4d). In this study, we found that HD-4d had hepatoprotective and anti-inflammatory effects on alcoholic liver injury in C57BL/6J mice, and it also had noticeable anti-inflammatory effects in EtOH and LPS-induced RAW264.7 cells. Besides, we found that HD-4d can reduce the expression of inflammatory factors by up-regulating NLRP12 in vivo and in vitro. We found that the expression of NLRP12 was significantly increased in EtOH and LPS-induced RAW264.7 cells compared with the control group. Moreover, the inhibitory effect of HD-4d on inflammation weakened considerably after silencing NLRP12 in RAW264.7 cells. However, when NLRP12 was overexpressed with plasmid pEX-3-NLRP12, the effect of HD-4d on alcohol and LPS induced inflammation was remarkably increased. In addition, further studies indicated that HD-4d inhibited the activation and phosphorylation of the p65 protein by up-regulating NLRP12. In conclusion, HD-4d activated NLRP12 to reduce liver injury and inflammatory response through the NF-кB pathway. [ABSTRACT FROM AUTHOR]

Dietary selenium putatively prevents oxidative damage, whereas excessive selenium may lead to animal disorder. In this study, we investigated the effects of low and excessive levels of dietary selenium on oxidative stress and mitochondrial proteins in mouse liver. Six to eight week old mice were fed a diet with low, excessive, or moderate (control) levels of selenium (sodium selenite). The selenium concentration and oxidative stress-related parameters in hepatic mitochondria were evaluated. Two-dimensional electrophoresis and mass spectrometry were applied to identify the differentially-expressed proteins associated with dietary selenium. The selenium content of the livers in mice with the low selenium diet was significantly lower than that of the control, while that of mice fed excessive levels was significantly higher. In both groups oxidative stress in hepatic mitochondria was found; accompanied by lower superoxide dismutase (SOD) and glutathione peroxidase (GPX) levels and higher malondialdehyde (MDA) content, compared with the control group. Furthermore, ten proteins in the hepatic mitochondria of the selenium-low or -excessive groups with more than two-fold differences in abundance compared with the control group were identified. The differentially-expressed proteins in hepatic mitochondria may be associated with dietary (low or excessive) selenium-induced oxidative stress. [ABSTRACT FROM AUTHOR]

Background: The purpose of this study is to investigate whether 18F-fluorodeoxyglucose (FDG) micro-positron emission tomography-computed tomography (microPET-CT) can be used to monitor a metabolic response to gefitinib in nude mouse tumor xenografts. Methods: Sixteen nude mice were implanted with human A431 epidermoid carcinoma cells and ten with human A549 lung adenocarcinoma cells, and the tumors were allowed to grow to an approximate size of 150 mm3. Ten and five of these mice, respectively, received intragastric gefitinib (100 mg/kg) once daily for 14 days, whereas six and five, respectively, received sterile water. Tumor metabolic activity was assessed by 18F-FDG microPET imaging before treatment (day 0) and on days 2, 7, and 14. Tumor uptake of 18F-FDG was determined from a region-of-interest drawn around the tumor, and the maximum percentage injected dose per gram (%ID/gmax) was calculated. Tumor volume measured on day 14 by microCT was used to categorize tumors as sensitive, stable, or resistant to gefitinib, and pathologic changes in these tumors were analyzed. Results: On day 2, the average changes in 18F-FDG uptake by A431 tumors sensitive, stable, and resistant to gefitinib were -30.92%±6.66%, -5.68% ± 6.95%, and 7.72%± 3.85%, respectively (P < 0.05 each), with no significant differences in the sizes of tumors sensitive and stable to gefitinib (P = 0.169). On day 7, sensitive tumors were significantly smaller than stable tumors (P = 0.034). On day 14, areas of necrosis were observed in gefitinib-sensitive tumors, with tumor necrosis ratios differing significantly among the sensitive, stable, and control groups (P < 0.05 each). In mice implanted with A549 cells, however, tumor 18F-FDG uptake, volume, and percent necrosis did not differ significantly between gefitinib-treated and untreated mice on days 0, 2, 7, and 14 (P > 0.05 each). Conclusions: F-FDG uptake is a sensitive method of detecting metabolic changes in tumors associated with therapy in vivo. [ABSTRACT FROM AUTHOR]

IL-22 is a cytokine that regulates tissue homeostasis at barrier surfaces. A variety of IL-22-producing cell types is known, but identification on the single-cell level remains difficult. Therefore, we generated a fate reporter mouse that would allow the identification of IL-22-producing cells and their fate mapping in vivo. To trace IL-22-expressing cells, a sequence encoding Cre recombinase was cloned into the Il22 locus, and IL22Cre mice were crossed with reporter mice expressing enhanced yellow fluorescence protein (eYFP) under control of the endogenous Rosa26 promoter. In IL22CreR26ReYFP mice, the fluorescent reporter permanently labels cells that have switched on Il22 expression, irrespective of cytokine production. Despite a degree of underreporting, eYFP expression was detectable in nonimmune mice and restricted to group 3 innate lymphoid cells (ILC3) in the gut and γδ T cells in skin or lung. Upon skin challenge with imiquimod, eYFP+ γδ and CD4 T cells expanded in the skin. Infection with Citrobacter rodentium initially was controlled by ILC3, followed by expansion of eYFP+ CD4 T cells, which were induced in innate lymphoid follicles in the colon. No eYFP expression was detected in small intestinal Th17 cells, and they did not expand in the immune response. Colonic eYFP+ CD4 T cells exhibited plasticity during infection with expression of additional cytokines, in contrast to ILC3, which remained largely stable. Single-cell quantitative PCR analysis of eYFP+ CD4 T cells confirmed their heterogeneity, suggesting that IL-22 expression is not confined to particular subsets or a dedicated Th22 subset. [ABSTRACT FROM AUTHOR]

Maternal nutrition may influence metabolic profiles in offspring. We aimed to investigate the effect of maternal folic acid supplement on glucose metabolism in mouse offspring fed a high-fat diet (HFD). Sixty C57BL/6 female mice were randomly assigned into three dietary groups and fed the AIN-93G diet containing 2 (control), 5 (recommended folic acid supplement, RFolS) or 40 (high folic acid supplement, HFolS) mg folic acid/kg of diet. All male offspring were fed HFD for eight weeks. Physiological, biochemical and genetic variables were measured. Before HFD feeding, developmental variables and metabolic profiles were comparable among each offspring group. However, after eight weeks of HFD feeding, the offspring of HFolS dams (Off-HFolS) were more vulnerable to suffer from obesity (p = 0.009), glucose intolerance (p < 0.001) and insulin resistance (p < 0.001), compared with the controls. Off-HFolS had reduced serum adiponectin concentration, accompanied with decreased adiponectin mRNA level but increased global DNA methylation level in white adipose tissue. In conclusion, our results suggest maternal HFolS exacerbates the detrimental effect of HFD on glucose intolerance and insulin resistance in male offspring, implying that HFolS during pregnancy should be adopted cautiously in the general population of pregnant women to avoid potential deleterious effect on the metabolic diseases in their offspring. [ABSTRACT FROM AUTHOR]

Elevated serum free fatty acids (FFAs) levels play an important role in the development of insulin resistance (IR) and diabetes. We investigated the dynamic changes and the underlying regulatory mechanism of postprandial FFA profile in hyperlipidemia (HLP) and their relation with insulin sensitivity in both humans and mice. We found that serum stearic acid (SA) is the only fatty acid that is increased dramatically in the postprandial state. The elevation of SA is due to increased insulin-stimulated de novo synthesis mediated by sterol regulatory element--binding protein-1c (SREBP-1c)/acetyl-CoA carboxylase/fatty acid synthase/elongation of long-chain fatty acid family member 6 (ELOVL6) and the elongation of palmitic acid (PA) catalyzed by ELOVL6. Downregulation of SREBP-1c or ELOVL6 by small interfering RNA can reduce SA synthesis in liver and serum SA level, followed by amelioration of IR in HLP mice. However, inhibition of SREBP-1c is more effective in improving IR than suppression of ELOVL6, which resulted in accumulation of PA. In summary, increased postprandial SA is caused by the insulin-stimulated SREBP-1c pathway and elongation of PA in HLP. Reduction of postprandial SA is a good candidate for improving IR, and SREBP-1c is potentially a better target to prevent IR and diabetes by decreasing SA. [ABSTRACT FROM AUTHOR]

The T-type Ca2+ channel (TTCC) plays important roles in cellular excitability and Ca2+ regulation. In the heart, TTCC is found in the sinoatrial nodal (SAN) and conduction cells. Cav3.1 encodes one of the three types of TTCCs. To date, there is no report regarding the regulation of Cav3.1 by β-adrenergic agonists, which is the topic of this study. Ventricular myocytes (VMs) from Cav3.1 double transgenic (TG) mice and SAN cells from wild type, Cav3.1 knockout, or Cav3.2 knockout mice were used to study β-adrenergic regulation of overexpressed or native Cav3.1-mediated T-type Ca2+ current (ICa-T(3.1)). ICa- T(3.1) was not found in control VMs but was robust in all examined TG-VMs. A β-adrenergic agonist (isoproterenol, ISO) and a cyclic AMP analog (dibutyryl-cAMP) significantly increased ICa-T(3.1) as well as ICa-L in TG-VMs at both physiological and room temperatures. The ISO effect on ICa-L and ICa-T in TG myocytes was blocked by H89, a PKA inhibitor. ICa-T was detected in control wildtype SAN cells but not in Cav3.1 knockout SAN cells, indicating the identity of ICa-T in normal SAN cells is mediated by Cav3.1. Real-time PCR confirmed the presence of Cav3.1 mRNA but not mRNAs of Cav3.2 and Cav3.3 in the SAN. ICa-T in SAN cells from wild type or Cav3.2 knockout mice was significantly increased by ISO, suggesting native Cav3.1 channels can be upregulated by the β-adrenergic (β-AR) system. In conclusion, β-adrenergic stimulation increases ICa-T(3.1) in cardiomyocytes, which is mediated by the cAMP/PKA pathway. The upregulation of ICa-T(3.1) by the β-adrenergic system could play important roles in cellular functions involving Cav3.1. [ABSTRACT FROM AUTHOR]

Background: Biomarkers facilitate early detection of disease and measurement of therapeutic efficacy, both at clinical and experimental levels. Recent advances in analytics and disease models allow comprehensive screening for biomarkers in complex diseases, such as asthma, that was previously not feasible. Objective: Using murine and nonhuman primate (NHP) models of asthma, identify biomarkers associated with early and chronic stages of asthma and responses to steroid treatment. Methods: The total protein content from thymic stromal lymphopoietin transgenic (TSLP Tg) mouse BAL fluid was ascertained by shotgun proteomics analysis. A subset of these potential markers was further analyzed in BAL fluid, BAL cell mRNA, and lung tissue mRNA during the stages of asthma and following corticosteroid treatment. Validation was conducted in murine and NHP models of allergic asthma. Results: Over 40 proteins were increased in the BAL fluid of TSLP Tg mice that were also detected by qRT-PCR in lung tissue and BAL cells, as well as in OVA-sensitive mice and house dust mite-sensitive NHP. Previously undescribed as asthma biomarkers, KLK1, Reg3γ, ITLN2, and LTF were modulated in asthmatic mice, and Clca3, Chi3l4 (YM2), and Ear11 were the first lung biomarkers to increase during disease and the last biomarkers to decline in response to therapy. In contrast, GP-39, LCN2, sICAM-1, YM1, Epx, Mmp12, and Klk1 were good indicators of early therapeutic intervention. In NHP, AMCase, sICAM-1, CLCA1, and GP-39 were reduced upon treatment with corticosteroids. Conclusions and clinical relevance: These results significantly advance our understanding of the biomarkers present in various tissue compartments in animal models of asthma, including those induced early during asthma and modulated with therapeutic intervention, and show that BAL cells (or their surrogate, induced sputum cells) are a viable choice for biomarker examination. [ABSTRACT FROM AUTHOR]

The aim of this study was to establish a murine model of lower genital tract infection by and and evaluate differences in pathogenicity of five serotypes. BALB/c female mice were divided into seven groups (five mice in each group), including five groups infected in the lower genital tract after treatment with estradiol with U. urealyticum serotypes 4 and 8 and U. parvum serotypes 1, 3, and 6, respectively, and two control groups of untreated mice and estradiol treated mice. The presence of infection was determined on solid and liquid culture media. Tumor necrosis factor-alpha (TNF-α) expression in lower genital tract secretions was determined by PCR, and morphological and histological changes of the lower genital tract were observed. The genital secretions of all inoculated mice were positive for U. urealyticum and U. parvum on culture in both liquid and solid media. TNF-α expression at 7 and 14 days after infection was markedly increased as compared with that of the controls. Morphological changes of the external genitalia included hair loss and erosions, and histological examination revealed infiltration by inflammatory cells. The five serotypes tested were all found to be pathogenic, and the pathogenicity varied with serotype 4 showing the greatest pathogenicity. [ABSTRACT FROM AUTHOR]

Background: The development of small molecule inhibitors of hepatitis C virus (HCV) core protein as antiviral agents has been intensively pursued as a viable strategy to eradicate HCV infection. However, lack of a robust and convenient small animal model has hampered the assessment of in vivo efficacy of any antiviral compound. Methodology/Principal Findings: The objective of this work was to develop a novel method to screen anti-core protein siRNA in the mouse liver by bioluminescence imaging. The inhibitory effect of two shRNAs targeting the highly conserved core region of the HCV genome, shRNA452 and shRNA523, was examined using this method. In the transient mouse model, the effect of shRNA-523 was detectable at as early as 24 h and became even more pronounced at later time points. The effect of shRNA-452 was not detectable until 48 h post-transduction. In a stable mouse model, shRNA523 reduced luciferase levels by up to 76.4±26.0% and 91.8±8.0% at 6 h and 12 h after injection respectively, and the inhibitory effect persisted for 1 day after a single injection while shRNA-Scramble did not seem to have an effect on the luciferase activity in vivo. Conclusions/Significance: Thus, we developed a simple and quantitative assay for real-time monitoring of HCV core protein inhibitors in mice. [ABSTRACT FROM AUTHOR]

Abstract: To find new liver-specific expression cassettes for long-term expression of therapeutic genes in the context of pDNA, the function and specificity of hepatitis B virus (HBV)’ two hepatic enhancers (EnI and EnII), combined with HBV core and X promoters in cultured cells were evaluated. By bioluminescence imaging and hydrodynamic gene transfer technology, the persistence of transgene expression containing these regulatory sequences in the liver of mice was assessed. Our data indicated that both HBV enhancers were able to stimulate HBV core and X promoter activity in cultured cells of hepatic origin. In vivo, HBV core promoter linked to EnI and EnII (EII–EI–Pc) and X promoter linked to EnI and EnII (EI–EII–Px) could direct a constant and high-level gene expression. [Copyright &y& Elsevier]

The kinetics of metabolic and inflammatory parameters associated with obesity were evaluated in a murine diet-induced obesity (DIO) model using a diet high in fat and cholesterol. Cellular infiltration and mediator production were assessed and shown to be therapeutically modulated by the PPARgamma agonist rosiglitazone. C57BL/6 mice were maintained on a 45% fat/ 0.12% cholesterol (HF/CH) or Chow diet for 3, 6, 16, or 27 weeks. Flow cytometry was employed tomonitor peripheral blood monocytes and adipose tissue macrophages (ATM). Gene expression and protein analysis methods were used to evaluate mediator production from total epididymal fat (EF), stromal vascular fraction (SVF), and sorted SVF cells. To investigate therapeutic intervention, mice were fed a HF/CH diet for 12 weeks and then a diet formulated with rosiglitazone (5mg/kg) for an additional 6 weeks. A HF/CH diet correlated with obesity and a dramatic proinflammatory state. Therapeutic intervention with rosiglitazone attenuated the HF/CH induced inflammation. In addition, a novel population was found that expressed the highest levels of the pro-inflammatory mediators CCL2 and IL-6. [ABSTRACT FROM AUTHOR]

We report here the development of a group of rabbit monoclonal antibodies against Smad1, Smad2, Smad3, and Smad5, and the immunocytochemistry (ICC) staining of human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). Eight New Zealand rabbits were immunized with synthesized peptides linked to KLH, and splenocytes from these rabbits were fused with rabbit immortal B cell 240E-W2. Resulting hybridomas producing anti-Smad monoclonal antibodies were screened by enzyme-linked immunosorbent assay (ELISA) with BSA-linked peptides. Clones were chosen for antibody production based on their activities in Western blotting and on paraffin-embedded human tissues, and the capacity of the antibodies in immunocytochemistry was demonstrated. Using these antibodies, we performed ICC staining on routinely cultured human and mouse embryonic stem cells, and showed that both cell types strongly express these genes. We propose that both hESCs and mESCs have the ability to transduce signals from both BMPs and TGFb/ Activin. [ABSTRACT FROM AUTHOR]

G-protein-coupled receptor (GPCR) 48 (Gpr48; Lgr4), a newly discovered member of the glycoprotein hormone receptor subfamily of GPCRs, is an orphan GPCR of unknown function. Using a knockout mouse model, we have characterized the essential roles of Gpr48 in bone formation and remodeling. Deletion of Gpr48 in mice results in a dramatic delay in osteoblast differentiation and mineralization, but not in chondrocyte proliferation and maturation, during embryonic bone formation. Postnatal bone remodeling is also significantly affected in Gpr48-/- mice, including the kinetic indices of bone formation rate, bone mineral density and osteoid formation, whereas the activity and number of osteoclasts are increased as assessed by tartrate-resistant acid phosphatase staining. Examination of the molecular mechanism of Gpr48 action in bone formation revealed that Gpr48 can activate the cAMP-PKA-CREB signaling pathway to regulate the expression level of Atf4 in osteoblasts. Furthermore, we show that Gpr48 significantly downregulates the expression levels of Atf4 target genes/proteins, such as osteocalcin (Ocn; Bglap2), bone sialoprotein (Bsp; Ibsp) and collagen. Together, our data demonstrate that Gpr48 regulates bone formation and remodeling through the cAMP-PKA-Atf4 signaling pathway. [ABSTRACT FROM AUTHOR]