Search

Wanqiu Zhu,1,* Xin Niu,1,* Mingxia Wang,1 Zhao Li,2 Hong-Kun Jiang,3 Chuntao Li,4 Samantha J Caton,5 Yinglong Bai11Department of Maternal and Child Health, School of Public Health, China Medical University, Shenyang, Liaoning, People’s Republic of China; 2Department of Cardiology, The First Hospital of China Medical University, Shenyang, Liaoning, People’s Republic of China; 3Department of Pediatrics, The First Hospital of China Medical University, Shenyang, Liaoning, People’s Republic of China; 4Information Center, the First Hospital of China Medical University, Shenyang, Liaoning, People’s Republic of China; 5School of Health and Related Research (ScHARR), Public Health, University of Sheffield, Sheffield, S1 4DA, UKCorrespondence: Yinglong BaiDepartment of Maternal and Child Health, School of Public Health, China Medical University, No.77 Puhe Road, Shenyang North New Area, Shenyang 110122, Liaoning, People’s Republic of ChinaTel+86 243 193 9406Fax +86 243 193 9406Email ylbai@cmu.edu.cn*These authors contributed equally to this workBackground: Consumption of industrially produced trans-fatty acids (iTFAs) can result in alteration to lipid profile and glucose metabolism. Moreover, a diet high in iTFAs could increase the risk of obesity, cardiovascular diseases (CVDs) and type 2 diabetes mellitus. Glucose and lipid metabolism are closely linked in white adipose tissue (WAT), yet the underlying mechanisms of the effect of iTFAs in WAT are poorly understood.Materials and methods: Parameters of glucose homeostasis, lipid profiles and markers of endoplasmic reticulum (ER) stress of WAT were measured in rats maintained on a high-fat diet containing margarine (HFD-M) (n=10) compared to controls maintained on standard chow (n=10) over 16 weeks.Results: Fat mass and body weight was significantly increased in rats maintained on the HFD-M compared to controls (P

Once thought to be non-naturally occurring, D-amino acids (DAAs) have in recent years been revealed to play a wide range of physiological roles across the tree of life, including in human systems. Synthetic biologists have since exploited DAAs' unique biophysical properties to generate peptides and proteins with novel or enhanced functions. However, while peptides and small proteins containing DAAs can be efficiently prepared in vitro , producing large-sized heterochiral proteins poses as a major challenge mainly due to absence of pre-existing DAA translational machinery and presence of endogenous chiral discriminators. Based on our previous work demonstrating pyrrolysyl-tRNA synthetase's (PylRS') remarkable substrate polyspecificity, this work attempts to increase PylRS' ability in directly charging tRNA Pyl with D-phenylalanine analogs (DFAs). We here report a novel, polyspecific Methanosarcina mazei PylRS mutant, DFRS2, capable of incorporating DFAs into proteins via ribosomal synthesis in vivo . To validate its utility, in vivo translational DAA substitution were performed in superfolder green fluorescent protein and human heavy chain ferritin, successfully altering both proteins' physiochemical properties. Furthermore, aminoacylation kinetic assays further demonstrated aminoacylation of DFAs by DFRS2 in vitro ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Jiang, Weng, Wang, Tsou, Chen, Ko, Söll, Tsai and Wang.)

Unique chemical and physical properties are introduced by inserting selenocysteine (Sec) at specific sites within proteins. Recombinant and facile production of eukaryotic selenoproteins would benefit from a yeast expression system; however, the selenoprotein biosynthetic pathway was lost in the evolution of the kingdom Fungi as it diverged from its eukaryotic relatives. Based on our previous development of efficient selenoprotein production in bacteria, we designed a novel Sec biosynthesis pathway in Saccharomyces cerevisiae using Aeromonas salmonicida translation components. S. cerevisiae tRNA Ser was mutated to resemble A. salmonicida tRNA Sec to allow recognition by S. cerevisiae seryl-tRNA synthetase as well as A. salmonicida selenocysteine synthase (SelA) and selenophosphate synthetase (SelD). Expression of these Sec pathway components was then combined with metabolic engineering of yeast to enable the production of active methionine sulfate reductase enzyme containing genetically encoded Sec. Our report is the first demonstration that yeast is capable of selenoprotein production by site-specific incorporation of Sec., (© 2023 Hoffman et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Purpose: The purpose of this study was to develop and validate a clinical predictive model for predicting the likelihood of a poor therapeutic response during the first year of recombinant human growth hormone (rhGH) treatment in children with growth disorders., Methods: A total of 627 pediatric patients with growth disorders (GHD, ISS, TS, SGA) from The LG Growth Study cohort were evaluated. Restricted cubic splines (RCS) were utilized to investigate the association between predictors and the risk of poor rhGH response. Variables were selected using LASSO regression, and multivariate logistics regression models were established. Receiver operating characteristic (ROC) curves, calibration curves, decision curve analysis (DCA), and clinical impact curves (CIC) were used to assess the predictive model's accuracy and clinical value. The predictive accuracy of the model was validated on the testing set., Results: Two predictive models containing 8 baseline predictors (diagnosis, age, height SDS, bone age minus chronological age, rhGH dosage, distance from mid-parental height in SDS, weight SDS, IGF-1 SDS) and 1 post-treatment predictor (height SDS gain at 6 months) were constructed by multivariate logistic regression analyses. The nomogram was built based on the multivariate predictive model and showed good discrimination and model fit effects in both the training set and the testing set. DCA and CIC analyses presented good clinical usability., Conclusion: The clinical predictive model for predicting the probability of poor short-term response of rhGH treatment in pediatric patients with growth disorders is useful and can assist physicians in making clinical decisions., (© 2022. The Author(s), under exclusive licence to Italian Society of Endocrinology (SIE).)

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. By activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in  Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein-protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

Multiple noncanonical amino acids can be installed into proteins in E. coli using mutually orthogonal aminoacyl-tRNA synthetase and tRNA pairs. Here we describe a protocol for simultaneously installing three distinct noncanonical amino acids into proteins for site-specific bioconjugation at three sites. This method relies on an engineered, UAU-suppressing, initiator tRNA, which is aminoacylated with a noncanonical amino acid by Methanocaldococcus jannaschii tyrosyl-tRNA synthetase. Using this initiator tRNA/aminoacyl-tRNA synthetase pair, together with the pyrrolysyl-tRNA synthetase/tRNA Pyl pairs from Methanosarcina mazei and Ca. Methanomethylophilus alvus, three noncanonical amino acids can be installed into proteins in response to the UAU, UAG, and UAA codons., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The pyrrolysyl-tRNA synthetase (PylRS) facilitates the cotranslational installation of the 22nd amino acid pyrrolysine. Owing to its tolerance for diverse amino acid substrates, and its orthogonality in multiple organisms, PylRS has emerged as a major route to install noncanonical amino acids into proteins in living cells. Recently, a novel class of PylRS enzymes was identified in a subset of methanogenic archaea. Enzymes within this class (ΔPylSn) lack the N-terminal tRNA-binding domain that is widely conserved amongst PylRS enzymes, yet remain active and orthogonal in bacteria and eukaryotes. In this study, we use biochemical and in vivo UAG-readthrough assays to characterize the aminoacylation efficiency and substrate spectrum of a ΔPylSn class PylRS from the archaeon Candidatus Methanomethylophilus alvus. We show that, compared with the full-length enzyme from Methanosarcina mazei, the Ca. M. alvus PylRS displays reduced aminoacylation efficiency but an expanded amino acid substrate spectrum. To gain insight into the evolution of ΔPylSn enzymes, we performed molecular phylogeny using 156 PylRS and 105 pyrrolysine tRNA (tRNA Pyl ) sequences from diverse archaea and bacteria. This analysis suggests that the PylRS•tRNA Pyl pair diverged before the evolution of the three domains of life, placing an early limit on the evolution of the Pyl-decoding trait. Furthermore, our results document the coevolutionary history of PylRS and tRNA Pyl and reveal the emergence of tRNA Pyl sequences with unique A73 and U73 discriminator bases. The orthogonality of these tRNA Pyl species with the more common G73-containing tRNA Pyl will enable future efforts to engineer PylRS systems for further genetic code expansion., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Short rib‑polydactyly syndrome type III (SRPS3) is a lethal perinatal skeletal disorder consisting of polydactyly and multi‑system organ abnormalities. To further assess the pathogenicity of two pairs of compound heterozygotes and to search for novel molecular etiology, X‑rays and hematoxylin and eosin staining were conducted in three cases: Two retrospective samples and a newly identified patient with SRPS3. In addition, next‑generation sequencing was used to evaluate a fetus with SRPS3. Typical radiological features of the three cases included a long, narrow thorax with short ribs, shortened long bones, spurs at the metaphysis of the long bones and congenital bowing of the femurs. The present study also observed atypical histopathological changes, together with the absence of proliferation and abundance of retaining cartilage in the primary spongiosum. In addition, two novel compound heterozygous variants were identified in the dynein cytoplasmic 2 heavy chain 1 ( DYNC2H1 ) gene of the fetus: NM_001080463.1, c.6591_6593delTGG (chr11:103055738‑103055740); NM_001080463.1, c.7883T>C (chr11:103070000). The findings of the present study provided further confirmation of the pathogenicity of two compound heterozygous variants in two retrospective samples and identified novel compound heterozygous variants. These findings may improve our knowledge of the histopathological and radiological changes in patients with SRPS3 and the relative effects of DYNC2H1 variants. The findings of the present study may facilitate the clinical and molecular diagnosis of SRPS3.

Ubiquitination is a versatile posttranslational modification that elicits signaling roles to impact on various cellular processes and disease states. The versatility is a result of the complexity of ubiquitin conjugates, ranging from a single ubiquitin monomer to polymers with different length and linkage types. Recent studies have revealed the abundant existence of branched ubiquitin chains in which one ubiquitin molecule is connected to two or more ubiquitin moieties in the same ubiquitin polymer. Compared to the homotypic ubiquitin chain, the branched chain is recognized or processed differently by readers and erasers of the ubiquitin system, respectively, resulting in a qualitative or quantitative alteration of the functional output. Furthermore, certain types of branched ubiquitination are induced by cellular stresses, implicating their important physiological role in stress adaption. In addition, the current chemical methodologies of solid phase peptide synthesis and expanding genetic code approach have been developed to synthesize different architectures of branched ubiquitin chains. The synthesized branched ubiquitin chains have shown their significance in understanding the topologies and binding partners of the branched chains. Here, we discuss the recent progresses on the detection, functional characterization and synthesis of branched ubiquitin chains as well as the future perspectives of this emerging field.

Ubiquitination and SUMOylation of protein are crucial for various biological responses. The recent unraveling of cross-talk between SUMO and ubiquitin (Ub) has shown the pressing needs to develop the platform for the synthesis of Ub tagged SUMO2 dimers to decipher its biological functions. Still, the platforms for facile synthesis of dimers under native condition are less explored and remain major challenges. Here, we have developed the platform that can expeditiously synthesize all eight Ub tagged SUMO2 and SUMOylated proteins under native condition. Expanding genetic code (EGC) method was employed to incorporate Se-alkylselenocysteine at lysine positions. Oxidative selenoxide elimination generates the electrophilic center, dehydroalanine, which upon Michael addition with C-terminal modified ubiquitin, a nucleophile, yield Ub tagged SUMO2. The dimers were further interrogated with USP7, a SUMO2 deubiquitinase, which is involved in DNA repair, to understand specificity toward the Ub tagged SUMO2 dimer. Our results have shown that the C-terminal domain of USP7 is crucial for USP7 efficiency and selectivity for the Ub tagged SUMO2 dimer.

Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA have been evolved to generate genetically encoded noncanonical amino acids (ncAAs). Use of tryptophan (Trp) analogues with pyrrole ring modification for their spatial and polarity tuning in enzyme activity and substrate specificity is still limited. Herein, we report the application of an evolved PylRS, FOWRS2, for efficient incorporation of five Trp analogues into the deubiquitinase USP30 to decipher the role of W475 for diubiquitin selectivity. Structures of the five FOWRS-C/Trp analogue complexes at 1.7-2.5 Å resolution showed multiple ncAA binding modes. The W475 near the USP30 active site was replaced with Trp analogues, and the effect on the activity as well as the selectivity toward diubiquitin linkage types was examined. It was found that the Trp analogue with a formyl group attached to the nitrogen atom of the indole ring led to an improved activity of USP30 likely due to enhanced polar interactions and that another Trp analogue, 3-benzothienyl-l-alanine, induced a unique K6-specificity. Collectively, genetically encoded noncanonical Trp analogues by evolved PylRS· tRNA CUA Pyl pair unravel the spatial role of USP30-W475 in its diubiquitin selectivity.

The Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)⋅tRNA Pyl pair can be used to incorporate non-canonical amino acids (ncAAs) into proteins at installed amber stop codons. Although engineering of the PylRS active site generates diverse binding pockets, the substrate ranges are found similar in charging lysine and phenylalanine analogs. To expand the diversity of the ncAA side chains that can be incorporated via the PylRS⋅tRNA Pyl pair, exploring remote interactions beyond the active site is an emerging approach in expanding the genetic code research. In this work, remote interactions between tRNA Pyl , the tRNA binding domain of PylRS, and/or an introduced non-structured linker between the N- and C-terminus of PylRS were studied. The substrate range of the PylRS⋅tRNA Pyl pair was visualized by producing sfGFP-UAG gene products, which also indicated amber suppression efficiencies and substrate specificity. The unstructured loop linking the N-terminal and C-terminal domains (CTDs) of PylRS has been suggested to regulate the interaction between PylRS and tRNA Pyl . In exploring the detailed role of the loop region, different lengths of the linker were inserted into the junction between the N-terminal and the C-terminal domains of PylRS to unearth the impact on remote effects. Our findings suggest that the insertion of a moderate-length linker tunes the interface between PylRS and tRNA Pyl and subsequently leads to improved suppression efficiencies. The suppression activity and the substrate specificity of PylRS were altered by introducing three mutations at or near the N-terminal domain of PylRS (N-PylRS). Using a N-PylRS⋅tRNA Pyl pair, three ncAA substrates, two S -benzyl cysteine and a histidine analog, were incorporated into the protein site specifically., (Copyright © 2020 Jiang, Lee, Tsou, Chang, Tseng, Chen, Li and Wang.)

Background: DNA methylation is a molecular modification of DNA that is vital and occurs in gene expression. In cancer tissues, the 5'-C-phosphate-G-3'(CpG) rich regions are abnormally hypermethylated or hypomethylated. Therefore, it is useful to find out the diseased CpG sites by employing specific methods. CpG sites are highly correlated with each other within the same gene or the same CpG island., Objective: Based on this group effect, we proposed an efficient and accurate method for selecting pathogenic CpG sites., Methods: Our method aimed to combine a L1/2 regularized solver and a central node fully connected network to penalize group constrained logistic regression model. Consequently, both sparsity and group effect were brought in with respect to the correlated regression coefficients., Results: Extensive simulation studies were used to compare our proposed approach with existing mainstream regularization in respect of classification accuracy and stability. The simulation results show that a greater predictive accuracy was attained in comparison to previous methods. Furthermore, our method was applied to over 20000 CpG sites and verified using the ovarian cancer data generated from Illumina Infinium HumanMethylation 27K Beadchip. In the result of the real dataset, not only the indicators of predictive accuracy are higher than the previous methods, but also more CpG sites containing genes are confirmed pathogenic. Additionally, the total number of CpG sites chosen is less than other methods and the results show higher accuracy rates in comparison to other methods in simulation and DNA methylation data., Conclusion: The proposed method offers an advanced tool to researchers in DNA methylation and can be a powerful tool for recognizing pathogenic CpG sites.

It is currently believed that the TBX1 gene is one of the core genes of congenital heart disease (CHD). However, there are few studies on the abnormal regulation of TBX1 gene expression. The purpose of this work was to investigate the role of miR-144 and TBX1 in cardiac development by studying the regulatory relationship and mechanism of miR-144 on TBX1/JAK2/STAT1 in cardiomyocytes. Cell proliferation was detected by MTT and clone formation assay and cell cycle and apoptosis by flow cytometry. The levels of miR-144 and TBX1 in H9c2 cells were assessed by qRT-PCR. Dual luciferase reporter assay was used to validate the direct targeting of TBX1 with miR-144. The protein expression levels of TBX1 and its downstream proteins were measured by Western blot analysis. miR-144 inhibited H9c2 cell proliferation by arresting cells in G1 phase. Furthermore, miR-144 induced H9c2 cell apoptosis and activated the JAK2/STAT1 signaling pathway. Bioinformatic predictions and luciferase reporter assay showed that miR-144 directly targets TBX1. Co-overexpression of miR-144 and TBX1 upregulated cell proliferation by accelerating G1 to S phase transition and downregulated cell apoptosis through inhibiting the JAK2/STAT1 signaling pathway. miR-144 acts as a proliferation inhibitor in cardiomyocytes via the TBX1/JAK2/STAT1 axis and is therefore a potential novel therapeutic target for CHD treatment., (© 2020 S. Karger AG, Basel.)

Methods based on a L 1/2 penalty have been utilized to solve the variable selection problem associated with the Cox proportional hazards model. One limitation of the existing methods for survival analysis is that these ignore the regulatory networks and pathways information. To merge prior pathway information into the analysis of genomic data, we proposed a network-based regularization method for the L 1/2 penalty and applied it to high-dimensional survival analysis data. This method used a L 1/2 regularized solver and network that penalizes a Cox proportional hazards model with respect to the sparsity of the regression and the smoothness between the coefficients in a given network. Based on the limited simulation studies and real breast cancer gene expression datasets, the experimental results showed that our method achieves a higher predictive accuracy than previous methods. Even though fewer genes were selected compared to those using previous methods, results showed stronger associations with cancer. The results of the analysis were also validated using GeneCards., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objectives: To investigate the effects of lipoxin A4 (LXA4) on inflammatory responses in obesity-related glomerulopathy (ORG) mouse model and its potential mechanisms., Methods: Male C57BL/6 mice were randomly divided into 4 groups: normal, model, LXA4, and LXA4/Boc-2 groups (n = 8). Mice in LXA4 group were intraperitoneally injected with LXA4 (40 ng/kg) once daily for 3 days following 12 weeks of high-fat diet (HFD) feeding. LXA4 receptor antagonist, Boc-2, was administered in LXA4/Boc-2 group prior to LXA4 treatment to block the effects of LXA4. Renal morphology and function impairment were determined. Inflammation was tested by measuring serum and mRNA levels of pro-inflammatory cytokines and chemokines. HFD-induced activation of nuclear factor-kappa B (NF-κB) and phosphorylation of mitogen-activated protein kinases (MAPKs) were investigated by immunohistochemistry and western blot., Results: HFD-feeding caused significant renal injury, pathological changes and inflammation in model group mice. LXA4 injection significantly alleviated HFD-induced effects on renal morphology and functions, as demonstrated by lower kidney index, glomerular diameter, 24 h urine protein, urinary albumin creatinine ratio and renal histomorphology. Moreover, HFD-induced accumulation of pro-inflammatory cytokines and chemokines were obviously attenuated by LXA4 administration, so did the HFD-induced activation of NF-κB and ERK/p38 MAPK pathways. However, these effects were markedly abrogated by BOC-2 pretreatment., Conclusion: LXA4 significantly attenuated HFD-induced renal inflammation and injury in ORG models, and these effects may be associated with the inhibition of activation of NF-κB and ERK/p38 MAPK pathways. The findings of our study may shed light on LXA4 showed a potential therapeutic application in ORG., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Viral nervous necrosis caused by nervous necrosis virus (NNV) is one of the most severe diseases resulting in high fish mortality rates and high economic losses in the giant grouper industry. Various NNV vaccines have been evaluated, such as inactivated viruses, virus-like particles (VLPs), recombinant coat proteins, synthetic peptides of coat proteins, and DNA vaccines. However, a cheaper manufacturing process and effective protection of NNV vaccines for commercial application are yet to be established. Hence, the present study developed a novel subunit vaccine composed of a carrier protein, receptor-binding domain of Pseudomonas exotoxin A, and tandem-repeated NNV coat protein epitopes by using the structural basis of epitope prediction and the linear array epitope (LAE) technique. On the basis of the crystal structure of the NNV coat protein, the epitope was predicted from the putative target cell receptor-binding region to elicit neutralizing immune responses. The safety of the LAE vaccine was evaluated, and all vaccinated fish survived without any physiological changes. The coat protein-specific antibody titers in the vaccinated fish increased after vaccine administration and exerted NNV-neutralizing effects. The efficacy tests revealed that the relative percent survival (RPS) of LAE antigen formulated with adjuvant was above 72% and LAE vaccine was effective for preventing NNV infection in giant grouper. This study is the first to develop an NNV vaccine by using epitope repeats, which provided effective protection to giant grouper against virus infection. The LAE construct can be used as a vaccine design platform against various pathogenic diseases., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Identifying biomarker genes and characterizing interaction pathways with high-dimensional and low-sample size microarray data is a major challenge in computational biology. In this field, the construction of protein-protein interaction (PPI) networks using disease-related selected genes has garnered much attention. Support vector machines (SVMs) are commonly used to classify patients, and a number of useful tools such as lasso, elastic net, SCAD, or other regularization methods can be combined with SVM models to select genes that are related to a disease. In the current study, we propose a new Net-SVM model that is different from other SVM models as it is combined with L1/2-norm regularization, which has good performance with high-dimensional and low-sample size microarray data for cancer classification, gene selection, and PPI network construction. Both simulation studies and real data experiments demonstrated that our proposed method outperformed other regularization methods such as lasso, SCAD, and elastic net. In conclusion, our model may help to select fewer but more relevant genes, and can be used to construct simple and informative PPI networks that are highly relevant to cancer., Competing Interests: The authors declare no conflict of interest.

Background: Hydrogen sulfide (H2S), a novel gaseous mediator, has been recognized as an important neuromodulator and neuroprotective agent in the nervous system. The present study was undertaken to study the effects of exogenous H2S on ischemia/reperfusion (I/R) injury of spinal cord and the underlying mechanisms., Methods: The effects of exogenous H2S on I/R injury were examined by using assessment of hind motor function, spinal cord infarct zone by Triphenyltetrazolium chloride (TTC) staining. Autophagy was evaluated by expressions of Microtubule associated protein 1 light chain 3 (LC3) and Beclin-1 which were determined by using Quantitative Real-Time PCR and Western blotting, respectively., Results: Compared to I/R injury groups, H2S pretreatment had reduced spinal cord infarct zone, improved hind motor function in rats. Quantitative Real-Time PCR or Western blotting results showed that H2S pretreatment also downregulated miR-30c expression and upregulated Beclin-1 and LC3II expression in spinal cord. In vitro, miR-30c was showed to exert negative effect on Beclin-1 expression by targeting its 3'UTR in SY-SH-5Y cells treated with Oxygen, Glucose Deprivation (OGD). In rat model of I/R injury, pretreatment of pre-miR-30c or 3-MA (an inhibitor for autophagy) can abrogated spinal cord protective effect of H2S., Conclusion: H2S protects spinal cord and induces autophagy via miR-30c in a rat model of spinal cord hemia-reperfusion injury.

Problems that screw cannot be inserted may occur in screw-rod fixation techniques such as Harms technique. We compared the biomechanical stability imparted to the C-2 vertebrae by four designed posterior screw and rod fixation techniques for the management of odontoid fractures. A three-dimensional finite element model of the odontoid fracture was established by subtracting several unit structures from the normal model from a healthy male volunteer. 4 different fixation techniques, shown as follows: ① C-1 lateral mass and C-2 pedicle screw fixation (Harms technique); ② C-1 lateral mass and unilateral C-2 pedicle screw fixation combined with ipsilateral laminar screw fixation; ③ Unilateral C-1lateral mass combined with ipsilateral C-1 posterior arch, and C-2 pedicle screw fixation; and ④ Unilateral C1 lateral mass screw connected with bilateral C2 pedicle screw fixation was performed on the odontoid fracture model. The model was validated for axial rotation, flexion, extension, lateral bending, and tension for 1.5 Nm. Changes in motion in flexion-extension, lateral bending, and axial rotation were calculated. The finite element model of the odontoid fracture was established in this paper. All of the four screw-rod techniques significantly decreased motion in flexion-extension, lateral bending, and axial rotation, as compared with the destabilized odontoid fracture complex (P<0.05). There was no statistically significant difference in stability among the four screw techniques. We concluded that the first three fixation techniques are recommended to be used as surgical intervention for odontoid fracture, while the last can be used as supplementary for the former three methods.

Previous findings have shown that acetylcholine (ACh) decreased hypoxia-induced tumor necrosis factor alpha (TNF α) production, thus protected against cardiomyocyte injury. However, whether and how ACh affects TNF α-induced endoplasmic reticulum (ER) stress and cell apoptosis remain poorly defined. This study was aimed at determining the effect of ACh in H9c2 cells after TNF α stimulation. Presence of ER stress was verified using the ER stress protein markers glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP). Cell apoptosis was shown by caspase-3 activation and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling. Exogenously administered ACh significantly decreased these TNF α-induced changes. Moreover, when the cells were exposed to nonspecific muscarinic receptor (M AChR) inhibitor atropine, methoctramine (M2 AChR inhibitor) or the epidermal growth factor receptor (EGFR) inhibitor AG1478, the cardioprotection elicited by ACh was diminished. Furthermore, the above effects were also blocked by M2 AChR or EGFR siRNA, indicating that EGFR transactivation by M2 AChR may be the major pathway responsible for the benefits of ACh. In addition, LY294002, a phosphatidylinositol-3-kinase (PI3K) inhibitor, displayed the similar trends as AG1478, suggesting that PI3K/Akt signaling may be the downstream of EGFR in ACh-elicited anti-apoptotic property. Together, these data indicate that EGFR-PI3K/Akt signaling is involved in M2 AChR-mediated ER apoptotic pathway suppression and the subsequent survival of H9c2 cardiomyocytes. We have identified a novel pathway underlying the cardioprotection afforded by ACh., (© 2014 Wiley Periodicals, Inc.)

Aerobic interval training (AIT) can favorably affect cardiovascular diseases. However, the effects of AIT on post-myocardial infarction (MI)-associated mitochondrial dysfunctions remain unclear. In this study, we investigated the protective effects of AIT on myocardial mitochondria in post-MI rats by focusing on mitochondrial dynamics (fusion and fission). Mitochondrial respiratory functions (as measured by the respiratory control ratio (RCR) and the ratio of ADP to oxygen consumption (P/O)); complex activities; dynamic proteins (mitofusin (mfn) 1/2, type 1 optic atrophy (OPA1) and dynamin-related protein1 (DRP1)); nuclear peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α); and the oxidative signaling of extracellular signal-regulated kinase (ERK) 1/2, c-Jun NH2-terminal protein kinase (JNK) and P53 were observed. Post-MI rats exhibited mitochondrial dysfunction and adverse mitochondrial network dynamics (reduced fusion and increased fission), which was associated with activated ERK1/2-JNK-P53 signaling and decreased nuclear PGC-1α. After AIT, MI-associated mitochondrial dysfunction was improved (elevated RCR and P/O and enhanced complex I, III and IV activities); in addition, increased fusion (mfn2 and OPA1), decreased fission (DRP1), elevated nuclear PGC-1α and inactivation of the ERK1/2-JNK-P53 signaling were observed. These data demonstrate that AIT may restore the post-MI mitochondrial function by inhibiting dynamics pathological remodeling, which may be associated with inactivation of ERK1/2-JNK-P53 signaling and increase in nuclear PGC-1α expression.

1. Aerobic interval training (AIT) exerts beneficial effects on cardiovascular disease. However, its cardioprotective mechanisms are not fully understood. The aim of the present study was to evaluate AIT-mediated anti-oxidation by focusing on anti-oxidase and mitochondrial biogenesis in rats after myocardial infarction (MI). 2. Sprague-Dawley rats were divided into three groups: (i) a sham-operated control (CON); (ii) an MI group; and (iii) an MI + AIT group. Myocardial microstructure and function, markers of oxidative stress, mitochondrial anti-oxidase, Phase II enzymes and mitochondrial biogenesis were assessed. In addition, levels of nuclear factor-erythroid 2-related factor (Nrf2) and phosphorylated (p-) AMP-activated protein kinase (AMPK) were determined. The anti-oxidative gene sirtuin 3 (SIRT3) and the prosurvival phosphatidylinositol-3 kinase (PI3-K)-protein kinase B (Akt) signalling cascade were also evaluated. 3. Compared with CON, there was noticeable microstructure injury, cardiac dysfunction and oxidative damage in rats after MI. In addition, decreased mitochondrial anti-oxidase content, Phase II enzyme (except heme oxygenase-1) expression and mitochondrial biogenesis were observed in the post-MI rats as well as reduced protein levels of the regulators Nrf2 and p-AMPK and suppression of SIRT3 levels and PI3-K/Akt signalling. These detrimental modifications were considerably ameliorated by AIT, as evidenced by increases in anti-oxidase, mitochondrial biogenesis, Nrf2 and AMPK phosphorylation, as well as SIRT3 upregulation and PI3-K/Akt signalling activation. Moreover, PI3-K inhibitor-LY294002 (20 mg/kg) treatment partly attenuated AIT-elicited increases in Nrf2 levels and AMPK phosphorylation. 4. Based on these results, we conclude that AIT effectively alleviates MI-induced oxidative injury, which may be closely correlated with activation of the anti-oxidase system and mitochondrial biosynthesis. Increased SIRT3 expression and activation of PI3-K/Akt signalling may play key roles in AIT-mediated anti-oxidation. These results open up new avenues for exercise intervention therapies for MI patients., (© 2014 Wiley Publishing Asia Pty Ltd.)

A double-chamber bioreactor has been developed to generate a tissue-engineered osteochondral composite (TEOC). However, a TEOC generally requires two types of cells (i.e. chondrogenic and osteogenic cells). Therefore, the capacity of mesenchymal stem cells (MSCs) as a single-cell source to work within a double-chamber bioreactor and biphasic scaffolds for generating TEOC was investigated. Compared with static culture, the double-chamber bioreactor not only can promote faster cellular proliferation, indicated by the PicoGreen dsDNA assay, SEM and confocal imaging, but also can trigger efficient chondrogenic and osteogenic differentiation of MSCs in biphasic scaffolds simultaneously, evidenced by gene expression. Thus MSCs are promising as the ideal single-cell source and the double-chamber bioreactor is an advanced culture system to generate TEOC.

1. Endothelial dysfunction plays a critical role in the development and progression or pathogenesis of hypertension. Amlodipine, a calcium channel blocker, is an effective antihypertensive agent. We investigated the effects of amlodipine on endothelial dysfunction in mesenteric arteries from spontaneously hypertensive rats (SHR). 2. Eight-week-old SHR were treated with amlodipine (10 mg/kg per day) for 8 weeks. Control SHR and Wistar-Kyoto (WKY) rats were treated with saline. Systolic blood pressure (SBP) was measured by the tail-cuff method. Isometric tension changes of isolated mesenteric arterial rings were recorded continuously by a myograph system. Serum contents of malondialdehyde (MDA) and total nitrate/nitrite (NO(x) ) were determined. Vascular superoxide anion production was analysed with dihydroethidium (DHE) fluorescence. 3. The contractile responses to KCl and phenylephrine were greater in untreated SHR than in WKY. Acetylcholine (ACh)-induced relaxation was significantly impaired in untreated SHR. Amlodipine treatment reduced the contractions and improved relaxation to ACh. In WKY, relaxation to ACh was inhibited by N(G) -nitro-l-arginine methyl ester (l-NAME) and not changed by ascorbic acid. In untreated SHR, the response to ACh was unaffected by l-NAME, whereas it was improved by ascorbic acid. Amlodipine restored the inhibitory effect of l-NAME on ACh-induced relaxation, but ascorbic acid no longer exerted its facilitating effect. Amlodipine prevented the rise in SBP and ameliorated abnormalities in serum MDA and NO in untreated SHR. DHE assay showed an increased intravascular superoxide generation in untreated SHR, which was abrogated by amlodipine. 4. Treatment of SHR with amlodipine resulted in amelioration of endothelial dysfunction by anti-oxidant activity and improvement in NO availability., (© 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.)

Background: The Cardiac α actin 1 gene (ACTC1) has been related to familial atrial septal defects. This study was set to explore a potential role of this gene in the formation of sporadic congenital heart disease (CHD)., Methods and Results: Assessment of cardiac tissue samples from 33 patients with sporadic CHD (gestational age (GA) 18 weeks-49 months) with real-time RT-PCR, Western blotting and immunohistochemistry has revealed a markedly decreased ACTC1 expression in the majority of samples (78.8%) compared with autopsied normal heart tissue from aged-matched subjects (GA 17 weeks-36 months). Also, as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, the proportion of apoptotic cardiomyocytes in samples featuring down-regulated ACTC1 expression (Group 1) was significantly greater than those with normal expression (Group 2) and the controls (P<0.01). The proportion of apoptotic cells strongly correlated with the expression of ACTC1 (r=-0.918, P<0.01). A study of 2 essential genes involved in apoptosis, Caspase-3 and Bcl-2, confirmed that the former has significantly increased expression, whilst the latter has decreased expression in Group 1 than in the other groups (P<0.01). Transfection of a small interfering RNA targeting, Actc1 (Actc1-siRNA), to a cardiomyocyte cell line, H9C2, also detected more apoptotic cells., Conclusions: Reduced ACTC1 expression might play a role in the onset of CHD through induction of cardiomyocyte apoptosis.

The aim of the present study was to investigate the alterations in thoracic aortic vasomotor function in rats with chronic heart failure (CHF) post myocardial infarction (MI), and then explored the possible mechanism of pathological changes. Male Sprague-Dawley rats were divided into sham and CHF groups randomly. The CHF model group of rats was generated by ligating the left anterior descending artery. In sham-operated rats the ligation was placed but not tightened. A total of 20 rats underwent either sham-operated (n=8) or surgery for MI (n=12). All sham-operated rats survived the surgical procedure and the post-surgical period, whereas total mortality among MI-rats was 25% (3 out of 12). Only MI-rats with infarct-size >30% of the left ventricle (LV) were included for analysis (8 out of 9). Ten weeks after surgery, rats were anaesthetized for hemodynamic measurements, which contains systolic pressure, diastolic pressure, left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), LV+dp/dt(max) and LV-dp/dt(max). After that hearts were rapidly excised and weighed. Myocardial infarct size was determined by triphenyltetrazolium chloride (TTC) staining method. Isolated thoracic artery ring preparations were studied in a wire-myograph. The arterial constrictive responses to KCl, CaCl2, phenylephrine (PE), and caffeine and the arterial diastolic responses to acetylcholine (ACh) were recorded by the Multi Myograph System. To explore the possible mechanism, nitric oxide synthase (NOS) inhibitor N-nitrl-L-arginine methylester (L-NAME) and non-selective cyclooxygenase (COX) inhibitor indomethacin (Indo) were used. The results obtained were as follows: (1) CHF group showed an increased contraction response to KCl (5-100 mmol/L) and PE (1x10(-8)-3x10(-4) mol/L), and a reduced endothelium-dependent relaxation response to ACh (1x10(-12)-1x10(-4) mol/L) compared with those observed in sham group (P<0.01, P<0.05); (2) In the presence of L-NAME (1 mmol/L), the endothelium-dependent cumulative contractions to ACh (1x10(-7)-1x 10(-4) mol/L) was significantly enhanced in CHF group (P<0.05), and this effect was reversed by pretreatment with Indo (10 mumol/L); (3) In CHF group, the vessels incubated with Indo (10 mumol/L) showed an increased vasodilation induced by ACh (1x10(-12)-1x10(-4) mol/L) (P<0.05); (4) In the Ca(2+)-free K-H solution, calcium-dependent contraction curves induced by CaCl2 (1x10(-4)-3x10(-2) mol/L) in CHF group significantly shifted to the left compared with sham group (P<0.05); while the vascular contraction induced by caffeine (30 mmol/L) had no significant changes. These findings suggest that thoracic arteries of rats with CHF have endothelial dysfunction, and the contribution of endothelial dilation and contraction was significantly altered in CHF rats. The mechanism could be partly associated with the increased endothelium-dependent contracting factors by COX pathway, or the increased extracellular Ca(2+) influx through voltage-operated channels, thus leading to elevated vasoconstriction.

Objective: To examine serum concentration of interleukin-18 (IL-18) and IL-18 mRNA expression in peripheral blood mononuclear cells (PBMCs) in children with primary nephrotic syndrome (PNS) and explore the possible role of IL-18 in steroid-resistant nephrotic syndrome (SRNS)., Methods: Sixty-six children with newly diagnosed PNS, including 39 cases of steroid sensitive nephrotic syndrome (SSNS) and 27 cases of SRNS, were enrolled. Forty healthy children were used as a normal control group. Blood samples were collected before and 8 weeks after glucocorticoid treatment. Serum concentration of IL-18 was measured using ELISA. IL-18 mRNA expression in PBMCs was detected by the RT-PCR method. The amount of 24-hr urine protein was measured by the biuret method. Serum contents of total cholesterol (T-Ch), triglyceride (TG), low density lipoprotein (LDL), total protein (TP), and albumin (Alb) were measured by the automatic biochemistry analyzer., Results: Serum concentration of IL-18 and IL-18 mRNA expression in PBMCs in the SSNS and the SRNS groups were significantly higher than those in the normal control group before treatment (P< 0.05). The SRNS group had increased serum protein concentration of IL-18 and IL-18 mRNA expression in PBMCs compared with the SSNS group before treatment (P< 0.05). Serum LDL content in the SRNS group was also significantly higher than that in the SSNS group before treatment (P< 0.05). After treatment, serum concentration of IL-18 and IL-18 mRNA expression in PBMCs in the SRNS group were significantly higher than those in the SSNS and the normal control groups (P< 0.05). Serum concentration of IL-18 and IL-18 mRNA expression in PBMCs in the SSNS group were significantly reduced after treatment, but the alterations of IL-18 were not observed in the SRNS group after treatment., Conclusions: SRNS was associated with increased serum IL-18 concentration and IL-18 mRNA expression in PBMCs. Over-production of IL-18 may play a role in the development of SRNS.

Objective: To study serum concentration and mRNA expression of interleukin-13 (IL-13) in children with steroid-responsive nephrotic syndrome (SRNS) and the effect of methylprednisolone pulse therapy (MPT) on IL-13 expression., Methods: Twenty-eight children with SRNS were enrolled in this study. Serum protein level of IL-13 was measured using ELISA and IL-13 mRNA expression in peripheral blood mononuclear cells (PBMC) was detected with RT-PCR before MPT, 2 and 5 days after MPT, and 2 weeks after disappearance of proteinuria following MPT. Twenty-four urinary protein was measured with the biuret assay. Twenty healthy children were used as controls., Results: Serum IL-13 levels (38.48 +/- 13.01 pg/mL vs 5.18 +/- 2.71 pg/mL) and PBMC IL-13 mRNA expression (1.31 +/- 0.23 vs 0.36 +/- 0.07) before MPT in SRNS patients were significantly higher than in the controls. After 5 days of MPT and 2 weeks after disappearance of proteinuria following MPT, serum IL-13 levels (15.33 +/- 7.81 and 5.35 +/- 2.12 pg/mL respectively) and PBMC IL-13 mRNA expression (0.89 +/- 0.26 and 0.33 +/- 0.08 respectively) were significantly reduced (P < 0.01). Serum IL-13 levels and PBMC IL-13 mRNA expression in SRNS patients 2 weeks after disappearance of proteinuria following MPT were reduced to control levels, but remained at a higher level than controls 5 days after MPT. A positive correlation was found between serum levels of IL-13 and 24-hour urinary protein in SRNS patients before (r=0.75, P < 0.01) and after 2 and 5 days of MPT (r=0.68, r=0.71 respectively; P < 0.05)., Conclusions: Serum IL-13 levels and PBMC IL-13 mRNA expression in children with SRNS increase. MPT can inhibit the expression of protein and mRNA of IL-13 in these patients.

Response of water quality to agricultural cultivation was investigated for the Liangwanghe River catchment of Fuxianhu Lake region in Yunnan Province of China. Two typical tillage lands-the rice-wheat rotation (R-W) and the tobacco-pea (T-P) rotation were selected and monitored. Groundwater quality and water quality of Liangwanghe River were monitored simultaneously, as well as the farmland cultivation situation at that time. It was found that cultivation activities, such as transplanting, base fertilizer applying, top dressing, draining would cause apparent elevation of concentrations of TP, PO4(3-)-P, TN, NO3(-)-N and NH4(+)-N of groundwater in a short time period. Nutrients adsorption by crops during different bearing periods has an obvious relationship to the change of contents of TP, PO4(3-)-P, TN, NO3(-)-N, and NH4(+)-N in groundwater in a long time scale. When the crops are in a peak demand for nutrients, contents of nutrients in groundwater were found to reduce obviously, and contents of nutrients may rise for other crop conditions. Contents of TP and PO4(3-)-P of Liangwanghe River for full cultivation periods were found to be 5.8% and 21.7% lower than those for partial cultivation periods, while contents of TN, NH4(+)-N and NO3(-)-N for full cultivation periods were found to be 11.5%, 242.6% and 9.55% higher than those for partial cultivation periods.

Objective Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a multifunctional protein that has thc capacity to modify cellular activities and to modulate matrix turnover. This paper revealed the contributive role of TIMP-1 in progressive muscular dystrophy (PMD). Methods We examined the expression and cellular localization of TIMP-1 protein using biopsied frozen muscle from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD) by immunohistochemistry, double immunofluorescence and Western blot analysis. Results The results of immunohistochemistry and double immunofluorescence showed that TIMP-1 was positive only in vascular endothelial cells of normal muscles. Immunohistochemistry and Western blot analysis showed that the staining intensity was distinctly increased in some dystrophic muscles of PMD for TIMP-1. Double immunofluorescence revealed that TIMP-1 strongly expressed in the regenerating muscle fibers, macrophages and macrophage infiltrating necrotic fibers. Some activated fibroblasts in endomysium and perimysium of DMD and CMD muscles were also positive for TIMP1. Conclusion The functional consequence of overexpression of TIMP-1 in the dystrophic muscles is unknown, but the elevated local expression of TIMP-1 in diseased muscles of PMD and their distinct distribution pattern provide evidence that TIMP-1 may participate in the pathogenesis of PMD.

Objective: Progressive muscular dystrophy (PMD) is characterized by muscle fiber necrosis, regeneration, and endomysial fibrosis. Although absence of dystrophin and subsarcolemmic protein has been known as the cause of muscle fiber degeneration, pathogenesis of interstitial fibrosis is still unknown. The aim of this study was to investigate the role of connective tissue growth factor (CTGF) in PMD and its relationship with muscular fibrosis., Methods: Immunological localization of CTGF was examined in frozen muscle specimens obtained via biopsy from 8 patients with Duchenne muscular dystrophy (DMD), 2 patients with Becker muscular dystrophy (BMD), 6 patients with congenital muscular dystrophy (CMD) and 6 cases with normal muscle by immunohistochemistry, double immunofluorescence and Western blot analysis., Results: The results of immunohistochemistry and double immunofluorescence showed that CTGF was positive only in vessels of normal muscle. Both immunohistochemistry and Western blot analysis showed that CTGF expression was distinctly increased in dystrophy muscles of PMD than that in normal muscles. In dystrophy muscle, marked immunostaining of CTGF was not only observed in vascular walls, but also strongly expressed in the cytoplasm and nuclei of regenerating muscle fibers, and also immunolocalized in the muscle fiber sarcolemma of non-regenerating fibers. Double labeling with antibodies against CTGF and CD68 demonstrated that CTGF was expressed in some macrophages and some macrophage infiltrated necrotic fibers. CTGF was strongly expressed in endomysial and perimysial connective tissues of dystrophy muscles of patients with DMD, CMD and FCMD. Double immunolabeling revealed that most activated fibroblasts in perimysium and endomysium were positive for CTGF, but not all of connective tissues were co-localized with CTGF. Older cases with FCMD showed poor or no expression of CTGF in advanced fibrosis., Conclusion: CTGF may play a role in the pathogenetic process of muscular dystrophy, and CTGF may be important for muscle repair and fibrosis.

While hypercortisolemia is commonly observed in depression, exactly where in the hypothalamic-pituitary-adrenocortical (H-P-A) axis this dysfunction arises remains undefined. In attempting to distinguish between central or peripheral locus of dysfunction, we studied in 12 patients (10 females, two males) with primary major depression and eight age-matched controls (six females, two males) in their adrenal cortisol response to infused adrenocorticotropic hormone (ACTH) (cosyntropin 0.05 microg/kg bodyweight) while endogenous ACTH was suppressed with 1 mg of dexamethasone. Compared with the control group, pre-dexamethasone plasma baseline cortisol level was significantly higher in depressed patients while ACTH level remained normal. Post-dexamethasone responses of both hormones were greatly non-suppressed in the depressed group. Exogenous cosyntropin-elicited rise in plasma cortisol was significantly lower in depressed patients while the ACTH response was not significantly different. These findings suggest that an adrenal cortisol response to ACTH was significantly decreased during depression as compared with normals in Chinese depressed patients. Therefore, the central mechanism of hyperfunctioning H-P-A axis causing hypercortisolemia should be emphasized.

Background: Patients with affective disorder often have a poorer than expected prognosis for functional outcome between episodes. Previously, these patients were thought to have a better outcome between episodes, particularly Chinese patients. The aim of our study was to prospectively monitor symptoms, and vocational and residential functions of a group of patients with bipolar affective disorder one year after hospitalization. This study compare these data with index, admission data and discuss the relationship between patients' symptoms and functional outcome., Methods: The Brief Psychiatric Rating Scale (BPRS), Modified Manic State Rating Scale (MMS) and Hamilton Depression Rating Scale (HDRS-21 items) were used to measure the patients' general psychiatric condition, manic and depressive symptoms, respectively. A modified Vocational State Index and modified Location Code Index Scale were used to determine levels of functioning., Results: One-year follow-up measurements indicated that 75% of patients were free of symptoms or had mild psychiatric symptoms and 95% of patients were asymptomatic or mildly symptomatic for affective-manic condition. There was significant improvement in symptom outcome. However, only 46% of patients were employed at the one-year follow-up assessment, and as few as 12% worked at their expected level of employment, while 42% were rated as being unable to work and remained at an incapacitated occupational function. There was a significant tendency for increased independent living at one year of follow-up (74.6% vs 45.8%). More than 40% of patients were solely responsible for themselves, while over 50% required others to be responsible for them in terms of daily living. Additionally, 70% of first-admission subjects were employed at some level at one-year follow-up assessment, compared with only 31.8% of subjects with multiple admissions; 70% of first-admission patients were living independently compared with only 40% of subjects with multiple admissions., Conclusions: The results showed that the symptom outcome was superior to the functional outcome. These findings indicate that variable factors other than psychotic symptoms are related to the functional outcome in Chinese patients with bipolar affective disorder. Although treatment reduced the symptom intensity of affective episodes, a long-range rehabilitative program and strategy for prevention relapse is required to help functional outcome catch up with symptomatic amelioration.

Adverse drug reactions of non-steroidal anti-inflammatory drugs (NSAIDs) are quite prevalent, but there are few reports about possible adverse psychiatric reactions, which may be ignored or underestimated. We describe here five psychiatric outpatients, two with major depressive disorders, one bipolar disorder, one schizophrenic disorder and one anxiety disorder, who were treated with NSAIDs for pain due to rheumatoid arthritis, osteoarthritis or other painful neuromuscular conditions. All five patients developed a moderate to severe depressive state, three patients became obviously paranoid, and four had either thoughts of suicide or an attempt while undergoing co-administration of NSAIDs. The psychiatric symptoms remitted when the NSAIDs were stopped. The depressive and paranoid symptoms returned on seven occasions of re-use or re-challenge with the same or a different type of NSAID in all five patients. When the NSAIDs were stopped again, the patients had another remission of the adverse psychiatric reactions, and eventually recovered to their baseline mental states in clear temporal relationships. The cases presented suggest that NSAIDs can induce or exacerbate idiosyncratic reproducible adverse psychiatric symptoms in certain vulnerable patients, including those with a variety of psychotic or neurotic disorders, and also in elderly persons, but these undesirable side-effects were generally transient and disappeared on withdrawal of the NSAIDs.

Although melatonin and/or cortisol secretions have been suggested as markers for both circadian and noradrenaline dysfunctions in psychiatric illnesses, especially in affective disorders, studies of melatonin and cortisol in schizophrenic patients are rare. We evaluated the circadian profiles of melatonin and cortisol secretion in schizophrenic patients and control subjects. A total of 21 medicated Taiwanese male paranoid schizophrenic inpatients (mean age, 27.3 +/- 7.2 yr) and 21 age- and sex-matched controls underwent 24-hour neuroendocrine screening. Melatonin and cortisol concentrations were measured at 2-hour intervals from 0800 h to 2200 h, and at 1-hour intervals from 2300 h to 0700 h. The standard dexamethasone suppression test was performed the next day to provide an index of hypothalamic-pituitary-adrenal axis (HPA) function. The results showed that the circadian rhythm of plasma melatonin secretion was disrupted in schizophrenics compared with controls, whereas the 24-hour profile of plasma cortisol was preserved. The melatonin to cortisol ratio was significantly higher in control subjects than in schizophrenic patients. Results of the dexamethasone suppression tests indicated that there were no functional changes in the HPA axis in schizophrenic patients. Five drug-naive schizophrenic patients studied simultaneously, but whose data were not included in the above analyses, had results consistent with those of the maintenance-medicated patients. Our findings suggest the presence of abnormal melatonin metabolism in Taiwanese schizophrenics, which may possibly be related to the pathophysiologic process itself. However, broader pathogenetic aspects of these neuroendocrine interrelations remain to be clarified.

Activation of the tyrosine hydroxylase (TH) gene in the adrenal medulla during stress is mediated by trans-synaptic mechanisms and may involve cholinergic receptors. Stimulation of nicotinic receptors in adrenal medullary cells induces cell depolarization, influx of Ca2+ ions and increases levels of cAMP. We have shown that both cAMP and membrane depolarization produce an increase in the expression of the TH gene in cultured bovine adrenal medullary cells (BAMC). Others have proposed that transcriptional activation of the TH gene by cAMP is mediated through the sequence homologous to a cAMP responsive element (CRE) located in the proximal region of the TH gene promoter. In the present study we have examined the mechanisms by which membrane depolarization increases the TH gene activity. Treatment of serum-free BAMC cultures with the depolarizing agent, veratridine, increased the extracellular concentration of catecholamines, Met5-enkephalin, and the relative abundance of TH mRNA. Veratridine treatment also increased the levels of mRNAs for the catecholamine biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT), and proenkephalin A (PEK). Treatment for longer than 3 h was required to increase TH mRNA levels. By contrast, our previous studies indicated that cAMP stimulation for 2 h produces a maximal increase in TH mRNA levels in BAMC. The effects of veratridine and forskolin on TH mRNA levels were additive, further indicating that depolarization and cAMP activate TH gene expression via different pathways. Calmidazolium, an antagonist of calmodulin, had no effect on the veratridine-induced increase in TH mRNA levels. Similarly sphingosine treatment or preincubation with PMA, which reduce protein kinase C (PKC) activity and attenuate the induction of TH mRNA by PMA or the hormone, angiotensin II, did not affect the induction by veratridine. To identify promoter mechanisms of TH gene activation in depolarized cells we transfected BAMC with a plasmid pTHgoodLuc and treated with veratridine for 24 h. pTHgoodLUC contains a luciferase reporter gene linked to a -428/+21 bp fragment of the bovine TH gene promoter (relative to the transcription start site). Veratridine increased the expression of luciferase from the TH promoter 2.5-fold. Deletion of the -194/-54 bp promoter region containing SP-1 and POU/Oct sites reduced veratridine stimulation by 40%. Additional deletion of the -269 to -190 bp promoter segment, including an AP-1 element, further reduced veratridine stimulation to a statistically non-significant level. In conclusion, activation of TH gene expression upon depolarization is not mediated by calmodulin and PKC. Promoter sequences involved in this activation are located upstream from the CRE. Depolarization may activate TH gene transcription by acting on more than one regulatory region.

The current study looked at plasma catecholamines, clinical autonomic function tests, and hemodynamic parameters in 10 ESRD patients (five men and five woman, aged 56.4 +/- 3.6) with dialysis hypotension and 10 patients (five men and five women, aged 58.6 +/- 4.2) without dialysis hypotension. Catecholamines were measured using high performance liquid chromatography--electrochemical detection (HPLC-ECD). Dialysis led to a significant decrease in mean arterial pressure (MAP) in the hypotensive group as compared with the normotensive group. Significantly higher basal (predialysis) plasma norepinephrine (NE) and dopamine levels (DA) were found in the hypotensive uremic group as compared with the normotensive group. Levels of plasma epinephrine (EP) were not significantly different between the normotensive and hypotensive groups. In response to postural stimulation, blood pressure fell in both groups, but the fall in the hypotensive group was significantly greater. Percentage increments of plasma catecholamines in response to postural stimulation in both groups were similar, however. Among the measured hemodynamic parameters, including total peripheral vascular resistance and left ventricular function (cardiac index and fractional shortening), only the cardiac index showed significantly lower values in the hypotensive group after dialysis, as compared with the normotensive group. Results of four tests of autonomic function indicated that although both groups responded similarly to hand-grip and cold-pressor tests, impaired responses to orthostasis and Valsalva maneuver after dialysis were observed in the hypotensive group. The MAP changes in dialysis in the hypotension prone group correlated inversely with predialysis plasma NE, but not with EP and DA.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of this study was to examine the effects of angiotensin on the release of enkephalin peptides and the expression of the proenkephalin (pEK) gene. Incubation of cultured bovine adrenal medullary (AM) cells in serum-free medium resulted in calcium- and time-dependent accumulation of [Met(5)]-enkephalin (MEK) in the medium. Fifteen minutes to three hours of incubation with 2 nM [Sar(1)]-angiotensin II (s(1)-AII) did not affect basal secretion of MEK; however, longer incubations (24 h) resulted in four- to fivefold increases. Northern and dot blot analyses with bovine pEK cDNA demonstrated increases in the relative abundance of pEK mRNA in angiotensin-treated cells, suggesting that the long-term increases in MEK release may reflect increased expression of pEK gene and MEK synthesis. Stimulation of MEK release and induction of pEK mRNA were concentration dependent (ED(50) approximately 1 nM. Changes in pEK mRNA levels were not observed until 12 h of incubation with s(1)-AII and continued to increase during an additional 12 h of incubation. Addition of an angiotensin antagonist, saralasin, at 0-16 h, but not at 18-20 h, to cells incubated continuously for 24 h with s(1)-AII inhibited changes in pEK mRNA and MEK release. These observations demonstrate the absence of apparent desensitization of angiotensin receptor function and indicate that long-term receptor-ligand interactions are required to induce changes in gene expression and MEK release. Induction of pEK mRNA and stimulation of MEK release were additive to the effects of veratridine and forskolin, respectively, indicating that the effects of angiotensin were not due to membrane depolarization or increased cyclic AMP levels. Angiotensin-induced increases in pEK mRNA were partially inhibited by nifedipine and also by dantrolene and TMB-8, drugs that inhibit voltage-dependent calcium channels and mobilization of calcium from intracellular stores, respectively. s(1)-AII-induced changes in pEK mRNA were inhibited with calmidazolium, suggesting involvement of calmodulin. The participation of protein kinase C in the induction of pEK gene and long-term stimulation of MEK release was indicated by inhibition of the s(1)-AII effects by pretreatment of cells with protein kinase C inhibitor sphingosine. Effects of s(1)-AII on induction of pEK mRNA by angiotensin and by nicotine were prevented by the translational inhibitor cycloheximide. In conclusion, angiotensin receptors were found to control expression of the pEK gene and secretion of MEK. Unlike nicotinic receptors, which may control secretion of enkephalin peptides directly by stimulating exocytosis and indirectly by controlling peptide synthesis, the effects of angiotensin appear to be mediated indirectly at the level of pEK gene expression.

Using selective neurotoxin lesioning and immunocytochemical techniques, the effects of dopamine receptor subtypes stimulation by selective subtype agonists in the modulation of striatal opioid dynorphin and enkephalin were characterized in the rat basal ganglia. Tissue staining with specific antibodies against either dynorphin A (1-17) or [Met5]-enkephalin was examined in the striatum and substantia nigra following unilateral nigrostriatal dopaminergic lesioning by the neurotoxin 6-hydroxydopamine (6-OHDA). Two weeks later three groups of five 6-OHDA lesioned rats were repeatedly treated with either the dopamine D-1 receptor agonist SKF-38393 (5 mg.kg, i.p.), D-2 receptor agonist LY-171555 (1 mg/kg, i.p.) or saline for 7 days and similar immunostaining procedures were employed. The results indicated that 6-OHDA lesioning alone led to reduction in dynorphin A (1-17) immunostaining in substantia nigra and striatum when compared to the sham-lesioned contralateral side. However, SKF-38393 treatment reversed that pattern and caused more intense dynorphin A (1-17) immunostaining than the lesion side, while LY-171555 had no effect. In contrast, enkephalin immunostaining was increased by 6-OHDA lesioning, and the effect was abolished by SKF-38393 but not LY-171555. These results are in agreement with previously reported radioimmunoassay findings and are compatible with the proposal that dopamine D-1 receptors mediate the excitatory effect on the dynorphinergic system while inhibiting the enkephalinergic system.

The purpose of this study was to obtain direct evidence that the nigrostriatal dopamine (DA) pathway modulates the metabolism of striatal dynorphin and [Met5]-enkephalin. This was achieved by repeated injections of apomorphine (APO) or D-amphetamine (AMP) in unilateral nigral 6-hydroxydopamine (6-OHDA)-lesioned rats. Three weeks after a 6-OHDA lesion, dynorphin A(1-8)-like immunoreactivity (DN-LI) and the level of mRNA encoding prodynorphin in the striatum on the lesioned side were decreased compared with the contralateral control side. Activation of DA receptors by 7 daily injections of APO (5 mg/kg, Bid, s.c.), however, caused a large increase (3- to 4-fold of saline control) in striatal levels of DN-LI and prodynorphin mRNA on the 6-OHDA lesioned side, which is far greater than the increase on the contralateral side (2-fold of saline control). Presumably, the potentiated effect of APO in 6-OHDA lesioned rats is due to hypersensitivity of DA receptors resulting from DA denervation. Seven daily injections of AMP (5 mg/kg, Bid, s.c.), a DA-releasing agent, increased striatal DN-LI (187% of saline control) on the non-lesioned side, but not on the 6-OHDA-lesioned side. Taken together, the data indicate that the nigrostriatal pathway exerts a tonic excitatory influence over the biosynthesis of dynorphin and that this influence is not maximal since an additional increase in dopaminergic tone further increases the expression of dynorphin. In contrast, [Met5]-enkephalin-like immunoreactivity (ME-LI) in the striatum was increased by a 6-OHDA-lesion (145% of contralateral control), which was blocked by repeated administration of APO but not AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of this study was to examine the effects of angiotensin on the enzyme activities and gene expression of two catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT), in bovine adrenal medullary (AM) cells. Short term (15 min) incubation of cultured AM cells with 2 nM [Sar1]angiotensin II (s1-AII) did not increase basal secretion of catecholamines; however, longer incubations (3, 24, or 72 h) produced 4-10-fold increases. To determine whether angiotensin affects synthesis of catecholamines, the activities of TH and PNMT were examined. Incubation with s1-AII (15-30 min) decreased the Km of TH for its biopterine cofactor [6R)-5,6,7,8-tetrahydro-1-biopterin dihydrochloride (BH4] without affecting the Vmax, suggesting activation of TH. After long term incubation (72 h) the Km value was identical to that of control, while increases in the apparent Vmax were observed. PNMT activity was unaffected during a 30-min treatment with s1-AII; however, 2-fold increases occurred after a 48-72-h incubation. s1-AII (24 h) increased the relative abundance of TH and PNMT mRNAs, suggesting that the long term increase in enzyme activities reflected increased expression of TH and PNMT genes. Maximal increases were observed at 2 nM s1-AII and the changes were antagonized by saralasin. Induction of TH mRNA by s1-AII was additive to the effects of veratridine or forskolin indicating that effects of angiotensin were not due to membrane depolarization or increased cyclic AMP levels. Incubation with Ca2+ ionophore A23187 increased TH and PNMT mRNA levels in AM cells raising the possibility that the increase in cellular [Ca2+] could mediate effects of angiotensin. Angiotensin-induced increases in TH and PNMT mRNA were inhibited by nifedipine indicating involvement of voltage-dependent Ca2+ channels. In addition, the increases in TH, but not PNMT mRNA, were antagonized by dantrolene, which inhibits mobilization of Ca2+ from intracellular stores. Calmodulin involvement was suggested by the inhibition of s1-AII induced changes in mRNA with 1 microM calmidazolium.(ABSTRACT TRUNCATED AT 400 WORDS)

Previous studies indicate that dopaminergic transmission inhibits the biosynthesis of enkephalin and stimulates that of dynorphin in the striatonigral pathway of intact rat. The purpose of this study was to determine which dopamine (DA) receptor subtype(s) mediate the modulatory actions of DA. We measured striatal and nigral levels of enkephalin and dynorphin in: (1) intact rats repeatedly injected with D1 (SKF-38393, 5 mg/kg, i.p.) or D2 (LY-171555, 1 mg/kg, i.p.) agonists, alone or in combination, (2) 6-hydroxydopamine (6-OHDA)-lesioned rats repeatedly injected with the same D1 or D2 agonists, and (3) intact rats repeatedly injected with D1 (SCH-23390, 0.05 mg/kg, s.c.) or D2 (sulpiride, 100 mg/kg, s.c.) antagonists, given alone or in combination with the mixed D1/D2 agonist apomorphine (5 mg/kg, i.p.). Repeated injections of the D1 agonist to intact rats (twice daily for 7 days) produced a small but not statistically significant increase in striatal levels of dynorphin; similar treatment with the D2 agonist did not affect dynorphin levels at all. Combined treatments with D1 and D2 agonists did not potentiate the effect of the D1 agonist. 6-OHDA lesions of the nigrostriatal DA pathway alone decreased the level of dynorphin in both the striatum and substantia nigra. However, repeated D1 agonist, but not D2, injections not only reversed the decrease in dynorphin levels, but caused a significant increase above control levels. In intact rats, repeated injections of the D1 or D2 antagonist alone failed to alter the levels of dynorphin, but the D1 antagonist, not the D2 antagonist, attenuated the apomorphine-induced increase in striatal dynorphin.(ABSTRACT TRUNCATED AT 250 WORDS)

The nuclear proto-oncogene, c-fos, has been implicated in the coordinated regulation of gene expression during cell proliferation and differentiation. In this study, we have demonstrated the induction of the c-fos gene products in differentiated cells of the adrenal medulla by non-mitogenic signals. Activation of adrenal medullary cells in vivo by insulin-induced hypoglycemia, and in vitro by nicotine or angiotensin resulted in the rapid and transient elevation of c-fos mRNA levels. Induction of the c-fos mRNA by angiotensin and nicotine were accompanied by the appearance of the c-fos protein. The increase in c-fos protein occurred initially in the cytoplasm and, later, in the nucleus, and it was co-localized with tyrosine hydroxylase. Nuclear expression of the c-fos protein was also induced by veratridine, forskolin and the calcium ionophore A231287. The role of calcium in the regulation of the c-fos gene by angiotensin with nifedipine and inhibition of the effects of angiotensin with nifedipine and sphingosine, a protein kinase C inhibitor. Activation of the c-fos gene may play a role in the coordinated induction of genes involved in the long-term adaptation of adrenal medullary cells to increased functional demands.

Adrenal responsiveness to Cosyntropin (synthetic ACTH) was investigated in five patients with major depression and five individually matched normal subjects. Three hours following suppression of endogenous ACTH secretion with dexamethasone (1 mg orally), the adrenal response to a 10-min infusion of Cosyntropin (0.05 micrograms/kg body weight) was monitored for 2 1/2 hr by plasma cortisol measured at 15-min intervals. The depressed patients had significantly higher baseline plasma cortisol, but not higher baseline ACTH, than the controls. During the 3-hr post-dexamethasone (and prior to Cosyntropin infusion), the depressed patients maintained significantly higher cortisol secretion, but not higher ACTH secretion, than the controls. After Cosyntropin infusion, there were no differences in ACTH and cortisol increases between the two groups. These findings stand in contrast to previous reports of enhanced adrenal responsiveness to the administration of much larger amounts of Cosyntropin in major depression.