COPI mediates retrograde trafficking from the Golgi to the endoplasmic reticulum (ER) and within the Golgi stack, sorting transmembrane proteins bearing C-terminal KKxx or KxKxx motifs. The structure of KxKxx motifs bound to the N-terminal WD-repeat domain of β'-COP identifies electrostatic contacts between the motif and complementary patches at the center of the β'-COP propeller. An absolute requirement of a two-residue spacing between the terminal carboxylate group and first lysine residue results from interactions of carbonyl groups in the motif backbone with basic side chains of β'-COP. Similar interactions are proposed to mediate binding of KKxx motifs by the homologous α-COP domain. Mutation of key interacting residues in either domain or in their cognate motifs abolishes in vitro binding and results in mistrafficking of dilysine-containing cargo in yeast without compromising cell viability. Flexibility between β'-COP WD-repeat domains and the location of cargo binding have implications for COPI coat assembly.

Relating the function of neuronal cell types to information processing and behavior is a central goal of neuroscience. In the hippocampus, pyramidal cells in CA1 and the subiculum process sensory and motor cues to form a cognitive map encoding spatial, contextual, and emotional information, which they transmit throughout the brain. Do these cells constitute a single class or are there multiple cell types with specialized functions? Using unbiased cluster analysis, we show that there are two morphologically and electrophysiologically distinct principal cell types that carry hippocampal output. We show further that these two cell types are inversely modulated by the synergistic action of glutamate and acetylcholine acting on metabotropic receptors that are central to hippocampal function. Combined with prior connectivity studies, our results support a model of hippocampal processing in which the two pyramidal cell types are predominantly segregated into two parallel pathways that process distinct modalities of information.

The prefrontal cortex (PFC) regulates emotional responses, but it is unclear how PFC integrates diverse inputs to select the appropriate response. We therefore evaluated the contribution of basolateral amygdala (BLA) and ventral hippocampus (vHPC) inputs to fear signaling in the prelimbic (PL) cortex, a PFC region critical for the expression of conditioned fear. In conditioned rats trained to press for food, BLA inactivation decreased the activity of projection cells in PL, and reduced PL conditioned tone responses. In contrast, vHPC inactivation decreased activity of interneurons in PL and increased PL conditioned tone responses. Consistent with hippocampal gating of fear after extinction, vHPC inactivation increased fear and PL pyramidal activity in extinguished, but not in conditioned, rats. These results suggest a prefrontal circuit whereby hippocampus gates amygdala-based fear. Thus, deficient hippocampal inhibition of PFC may underlie emotional disorders, especially in light of reduced hippocampal volume observed in depression and PTSD.

The aim of the present study was to evaluate the hepatoprotective activity of lantadene A against acetaminophen-induced liver toxicity in mice was studied. Activity was measured by monitoring the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and bilirubin, along with histo-pathological analysis. Silymarin was used as positive control. A bimodal pattern of behavioural toxicity was exhibited by the lantadene A-treated group at the beginning of the treatment. However, treatment with lantadene A and silymarin resulted in an increase in the liver weight compared with the acetaminophen treated group. The results of the acetaminophen-induced liver toxicity experiments showed that mice treated with lantadene A (500 mg/kg) showed a significant decrease in the activity of ALT, AST and ALP and the level of bilirubin, which were all elevated in the acetaminophen treated group (p < 0.05). Histological studies supported the biochemical findings and a maximum improvement in the histoarchitecture was seen. The lantadene A-treated group showed remarkable protective effects against histopathological alterations, with comparable results to the silymarin treated group. The current study confirmed the hepatoprotective effects of lantadene A against the model hepatotoxicant acetaminophen, which is likely related to its potent antioxidative activity.

NANOG, OCT4 and SOX2 form the core network of transcription factors supporting embryonic stem (ES) cell self-renewal. While OCT4 and SOX2 expression is relatively uniform, ES cells fluctuate between states of high NANOG expression possessing high self-renewal efficiency, and low NANOG expression exhibiting increased differentiation propensity. NANOG, OCT4 and SOX2 are currently considered to activate transcription of each of the three genes, an architecture that cannot readily account for NANOG heterogeneity. Here, we examine the architecture of the Nanog-centred network using inducible NANOG gain- and loss-of-function approaches. Rather than activating itself, Nanog activity is autorepressive and OCT4/SOX2-independent. Moreover, the influence of Nanog on Oct4 and Sox2 expression is minimal. Using Nanog:GFP reporters, we show that Nanog autorepression is a major regulator of Nanog transcription switching. We conclude that the architecture of the pluripotency gene regulatory network encodes the capacity to generate reversible states of Nanog transcription via a Nanog-centred autorepressive loop. Therefore, cellular variability in self-renewal efficiency is an emergent property of the pluripotency gene regulatory network.

To investigate the growth potential of keratinocytes derived from the germinative epithelium (GE) of ovine hair follicles. Stem cells from the outer root sheath (ORS) of hair follicles migrate to the GE in the lower follicle where they proliferate and differentiate to form the hair fiber. It has been suggested that the GE comprises transit-amplifying cells and that the duration of anagen is determined by their limited proliferative potential. However, we show here that keratinocytes derived from the GE of ovine follicles grow extensively in vitro, arguing against this hypothesis.

We investigated how musical phrasing and motor sequencing interact to yield timing patterns in the conservatory students' playing piano scales. We propose a novel analysis method that compared the measured note onsets to an objectively regular scale fitted to the data. Subsequently, we segment the timing variability into (i) systematic deviations from objective evenness that are perhaps residuals of expressive timing or of perceptual biases and (ii) non-systematic deviations that can be interpreted as motor execution errors, perhaps due to noise in the nervous system. The former, systematic deviations reveal that the two-octave scales are played as a single musical phrase. The latter, trial-to-trial variabilities reveal that pianists' timing was less consistent at the boundaries between the octaves, providing evidence that the octave is represented as a single motor sequence. These effects cannot be explained by low-level properties of the motor task such as the thumb passage and also did not show up in simulated scales with temporal jitter. Intriguingly, this instability in motor production around the octave boundary is mirrored by an impairment in the detection of timing deviations at those positions, suggesting that chunks overlap between perception and action. We conclude that the octave boundary instability in the scale playing motor program provides behavioral evidence that our brain chunks musical sequences into octave units that do not coincide with musical phrases. Our results indicate that trial-to-trial variability is a novel and meaningful indicator of this chunking. The procedure can readily be extended to a variety of tasks to help understand how movements are divided into units and what processing occurs at their boundaries.

Astroglial cells possess an array of cellular defense systems, including superoxide dismutase (SOD) and catalase antioxidant enzymes, to prevent damage caused by oxidative stress on the central nervous system. Astrocytes specifically synthesize and release endozepines, a family of regulatory peptides including the octadecaneuropeptide (ODN). ODN is the ligand of both central-type benzodiazepine receptors (CBR), and an adenylyl cyclase- and phospholipase C-coupled receptor. We have recently shown that ODN is a potent protective agent that prevents hydrogen peroxide (H(2)O(2))-induced inhibition of SOD and catalase activities and stimulation of cell apoptosis in astrocytes. The purpose of the present study was to investigate the type of receptor involved in ODN-induced inhibition of SOD and catalase in cultured rat astrocytes. We found that ODN induced a rapid stimulation of SOD and catalase gene transcription in a concentration-dependent manner. In addition, 0.1 nM ODN blocked H(2)O(2)-evoked reduction of both mRNA levels and activities of SOD and catalase. Furthermore, the inhibitory actions of ODN on the deleterious effects of H(2)O(2) on SOD and catalase were abrogated by the metabotropic ODN receptor antagonist cyclo(1-8)[Dleu(5)]OP, but not by the CBR antagonist flumazenil. Finally, the protective action of ODN against H(2)O(2)-evoked inhibition of endogenous antioxidant systems in astrocytes was protein kinase A (PKA)-dependent, but protein kinase C-independent. Taken together, these data demonstrate for the first time that ODN, acting through its metabotropic receptor coupled to the PKA pathway, prevents oxidative stress-induced alteration of antioxidant enzyme expression and activities. The peptide ODN is thus a potential candidate for the development of specific agonists that would selectively mimic its protective activity.

New, in silico ways of generating hypotheses based on large data sets have emerged in the past decade. These data sets have been used to investigate different aspects of plant biology, especially at the level of transcriptome, from tissue-specific expression patterns to patterns in as little as a few cells. Such publicly available data are a boon to researchers for hypothesis generation by providing a guide for experimental work such as phenotyping or genetic analysis. More advanced computational methods can leverage these data via gene coexpression analysis, the results of which can be visualized and refined using network analysis. Other kinds of networks of, e.g., protein-protein interactions, can also be used to inform biology. These networks can be visualized and analyzed with additional information on gene expression levels, subcellular localization, etc., or with other emerging kinds information. Finally, cross-level correlation is an area that will become increasingly important. Visualizing these cross-level correlations will require new data visualization tools.

FRAMESHIFTING RESULTS FROM TWO MAIN MECHANISMS: genomic insertions or deletions (indels) or programmed ribosomal frameshifting. Whereas indels can disrupt normal protein function, programmed ribosomal frameshifting can result in dual-coding genes, each of which can produce multiple functional products. Here, I summarize technical advances that have made it possible to identify programmed ribosomal frameshifting events in a systematic way. The results of these studies suggest that such frameshifting occurs in all genomes, and I will discuss methods that could help characterize the resulting alternative proteomes.

Royan B(1) stem cell can be differentiated to specialized cell types including cardiomyocytes. This developmental change is accompanied with expression of various K(+) channel types. The aim of this study was to detect functional expression of K(+) currents from stem cell stage and one week and two weeks after differentiation into cardiomyocyte. Mouse stem cell derived cardiomyocytes (ES-cardiomyocytes) were isolated to single cell suspension for K(+) current recording using whole cell patch-clamp technique. The predominant depolarizing current in ES-cardiomyocytes was a tetraethylammonium (TEA) (10 mM) sensitive current which was partially blocked by nifedipine (1 μM) and attenuated by increasing concentration of EGTA (10 mM) in the pipette solution. Pharmacology and electrophysiological properties of this oscillatory sustained current very well matched with characteristics of Ca(2+) activated K(+) current. In addition there was another kind of sustained outward K(+) current which was resistance to TEA but was inhibited by 3,4-diaminopyridine. The characteristic features of this current indicate that this current was due to activation of delayed rectifier K(+) channels. RT-PCR study also confirmed expression of these two types of K(+) channels in ES-cardiomyocytes. Therefore, present study shows functional expression of two types of K(+) ionic current in ES-cardiomyocytes.

Carum copticum from Apiaceae family has several biological effects including analgesic, anti-inflammatory, anxiolytic and antispasmodic activities. This study was designed to evaluate its effect on suppression of naloxone-induced morphine withdrawal signs. Hydroalcoholic and polyphenolic extracts were prepared according to the standard methods. Male mice (25-30 g) were made dependent by subcutaneous injection of increasing doses (30-90 mg/kg) of morphine. Withdrawal syndrome was elicited by naloxone (5 mg/kg, i.p.) and number of jumpings and also presence of ptosis, hyperventilation, piloerection, tremor and diarrhea were evaluated during a 30 min period started immediately after naloxone injection. The hydroalcoholic extract at doses of 1 and 2 g/kg and the polyphenolic extract at doses of 0.5, 1 and 2 g/kg significantly (P<0.05) inhibited jumpings. Both extracts inhibited tremor significantly (P<0.01). Also the maximum applied dose of the extracts significantly (P<0.05) reduced ptosis. These results clearly show that Carum copticum is effective in suppression of morphine withdrawal and further studies are needed to find out the responsible constituents and also the mechanism of their actions.

Several species of Prangos are traditionally used as emollient, carminative, tonic, anti-flatulent, anthelmintic and anti-thrombotic agents. Osthole, a coumarin isolated from Prangos, are believed to be responsible for some of its effects. In this research, relaxant effects of Prangos ferulacea extract and osthole on rat uterus contraction induced by KCl, acetylcholine (ACh), oxytocin and electrical field stimulation (EFS) was investigated and compared with atropine and salbutamol. P. ferulacea acetonic extract concentration-dependently relaxed uterine contraction induced by KCl (IC(50)=13 ± 0.81 μg/ml), ACh (IC(50)=12 ± 1.38 μg/ml), oxytocin (IC(50)=16 ± 3.14 μg/ml) and EFS (IC(50)=11 ± 1.5 μg/ml). However, the extract at lower concentration (2.5 μg/ml) potentiated the EFS response. Osthole only had inhibitory effect on rat uterus and its relaxant effect was observed at lower concentration in comparison with P. ferulacea extract. Osthole in a similar way inhibited the response to KCl (IC(50)=4 ± 0.13 μg/ml), ACh (IC(50)=4 ± 0.8 μg/ml), oxytocin (IC(50)=4 ± 0.8 μg/ml) and EFS (IC(50)=1.5 ± 0.5 μg/ml). Our results demonstrated that osthole acted directly on uterus smooth muscle to induce relaxation, whereas P. ferulacea caused both contraction and relaxation of rat uterine smooth muscle. The relaxation of osthole might be mediated through Ca(2+) channel blocking activity as it inhibited the response to KCl. Mechanisms other than Ca(2+) channel blocking appeared to be responsible for ACh relaxation effect of osthole.

The protein-bound polysaccharide K (PSK) is used as a non-specific immunotherapeutic agent for the treatment of colon cancer. Little research, however, has been conducted on its association with angiogenesis, which is a prognostic factor markedly correlated with hematogenous metastases. We therefore decided to investigate the action of PSK on angiogenic growth factors, angiogenesis inhibitors and angiogenesis in colon cancer cells. Reverse transcription-polymerase chain reaction (RT-PCR) was used to investigate changes in HIF-1α mRNA expression. PCR array was used to investigate changes in angiogenic growth factors and angiogenesis inhibitors, as well as the expression of related genes. Colon cancer cells were cultured with or without PSK for 48 h. The following day, cells were cultured for two days at 37°C in new complete media. The resulting culture medium was placed in the chamber of a tube formation system in order to investigate tube formation. Investigation of HIF-1α mRNA expression in colon cancer cell lines and in cells cultured under identical conditions with added PSK revealed a significant decrease in expression, as well as a decrease in angiogenic growth factors and related genes in PSK-treated colon cancer cell lines. By contrast, levels of angiogenesis inhibitors and related genes were higher in the PSK-treated colon cancer cell lines. Investigation of tube formation revealed that elongation was inhibited in the medium of the PSK-treated colon cancer cell lines in comparison to the medium of the non-treated colon cancer cell lines. PSK suppresses angiogenic growth factors and related genes, enhances angiogenesis inhibitors and related genes and ultimately suppresses angiogenesis in colon cancer cells.

The present study aimed to observe the effect of rat bone marrow mesenchymal stem cells (MSCs) in vitro on hepatic stellate cell (HSC) RhoA signaling factors and the expression of the cell cycle regulators P27 and cyclin D1. Rat HSC-T6 and fibroblast cells were divided into control, negative control and MSC experimental groups. The cell proliferation rate was examined using the WST8 assay. The cell cycle was analyzed using flow cytometry. RT-PCR and western blot analysis were used to examine cyclin in D1 (cyclin D1), RhoA and P27 mRNA and protein expression in HSCs. After 12 h of co-culture, transition of the MSCs from the G0/G1 to S phase was blocked by HSCs. In the MSC experimental group, the RhoA mRNA and RhoA protein expression showed a decreasing trend with time, which was statistically significant compared with that in the control and negative control groups. MSC P27 protein expression showed an increasing trend with time. RhoA and P27 expression were significantly negatively correlated. After 24 h of co-culture, MSCs inhibited cyclin D1 expression. The difference was statistically significant in the experimental and control groups as well as in the negative control group (P<0.01). In conclusion, co-culture of HSCs with MSCs is capable of inhibiting HSC proliferation, promoting apoptosis and inhibiting RhoA expression. Reduced RhoA activity may induce an upregulation in P27 protein expression in HSCs, which promotes the inhibition of cyclin D1 by MSCs and induces cell cycle arrest at the G0/G1 phase, indicating a role in inhibiting rat HSC proliferation.

The aim of the present study was to create a feasible specific rat model of isolated bilateral pulmonary contusion (PC) and to evaluate the relationship between severity of hypoxemia and quantity of contusion lesions. Anesthetized rats were placed in a prone position. Injury energy ranging from 2.1 to 3.0 J was produced by a falling weight passed through a specially designed arched shield to the bilateral chest wall of rats. After injury (4 h), the contusion volume was measured using computer-generated three-dimensional reconstruction from a chest computed tomographic scan and expressed as a percentage of total lung volume. Arterial partial pressure of oxygen (PaO(2)) in blood gas analysis and contusion volume percentage were used to assess the severity of contusion. Heart and lung biopsy was used to confirm the diagnosis and rule out the existence of myocardial contusion. There were 3 cases of death and 1 case of death in the 3.0 J and the 2.4 J group, respectively. PaO(2) in the 2.7 J group was significantly lower than that in the lower energy groups (P<0.001). The percentage of pulmonary contusion in the 2.7 J group was significantly higher compared to that of the lower energy groups (P<0.001). PaO(2) was negatively correlated with contusion percentage (R(2)=0.76). Hemorrhage, edema and neutrophil infiltration were determined by lung biopsy. No evidence of myocardial contusion was documented in multiple heart biopsies. The method illustrated in this research effectively duplicates isolated bilateral pulmonary contusion in rats, the severity of which is highly correlated with the contusion size. Thus, 2.7 J can be regarded as the maximal energy for sublethal injury.

Identifying prognostic factors for osteosarcoma (OS) aids in the selection of patients who require more aggressive management. CD133 has been found to be a prognostic factor of certain tumor types. However, the association between CD133 expression and the prognosis of OS remains unknown. In this study, we analyzed the association of CD133 expression in OS with clinical factors and overall survival, and further investigated its potential role in metastasis in vitro. We found CD133 expression in 65.7% (46/70) of OS samples using immunohistochemistry, and it was positively correlated with lung metastasis analyzed by Chi-square test (P=0.002) and shorter overall survival time using the Kaplan-Meier method compared by log-rank test (P=0.000). Multivariate analysis showed that CD133 expression was an independent prognostic factor of patients with OS. To test for direct participation of CD133, we separated CD133(+) and CD133(-) cells in the MG63 cell line using magnetic-activated cell sorting and found that CD133(+) cells were more active in migration by scratch wound-healing assay and invasion by Matrigel invasion assay compared with CD133(-) cells. Elevated mRNA expression of the stemness gene octamer-binding transcription factor 4 (Oct-4) and NANOG, and the metastasis-related receptor C-X-C chemokine receptor type 4 (CXCR4) were also found in CD133(+) cells by reverse transcription-polymerase chain reaction. Thus, expression of CD133 was correlated with lung metastasis and poor prognosis in OS patients. CD133(+) cells may be a type of cancer stem cell with high expression of self-renewal capacity and metastasis-related genes.

Obesity and β-cell dysfunction due to oxidative stress impact the pathogenesis of type 2 diabetes mellitus. We co-cultured 3T3L1 adipocytes and islet cells in the presence or absence of the antioxidant α-lipoic acid (LA) and assayed the effects of the adipocytes and LA on the secretion of insulin by the islet cells and on the activities of factors involved in secretion and oxidative stress. At low glucose concentrations (2.8 mmol/l), the presence of adipocytes (co-culture) increased insulin secretion compared with islet cells cultured alone (control) and this increase was diminished by LA (co-culture plus LA). At high glucose concentrations (22 mmol/l), insulin secretion levels were similar for all islet groups, resulting in a restoration of the stimulation index in the presence of LA. The mRNA levels of the glucose-stimulated insulin secretion (GSIS) genes glucokinase, glucose transporter 2 and Kir6.2 were downregulated under co-culture and co-culture plus LA conditions. Protein and tyrosine phosphorylation levels of insulin receptor-β and insulin receptor substrate-1 were decreased under co-culture conditions and were restored by LA treatment. Cellular malondialdehyde levels increased in the co-cultured islets and this increase was blocked by LA. The mRNA levels of superoxide dismutase and catalase were reduced under co-culture conditions and these reductions were eliminated by the addition of LA. In conclusion, 3T3L1 adipocytes disturb insulin secretion and induce islet dysfunction. The effects may be mediated by multiple pathways, which include downregulation of GSIS gene expression, suppression of islet cell insulin signaling and the induction of oxidative stress. LA may protect islet cells via activation of islet cell insulin signaling and the mRNA expression of antioxidant enzymes.

In the development of anti-blood cancer drugs, the chronic myelocytic leukemia (KU812), acute myelocytic leukemia (KG-1) and lymphoma (U937) cell lines are commonly used in preclinical pharmacology studies as human cancer xenograft models in mice. In the present study, mRNA expression levels of typical human ATP-binding cassette (ABC) transporters in these human blood cancer cell lines were analyzed by real-time polymerase chain reaction (RT-PCR). Based on the results, the expression level of multidrug resistance-associated protein 4 (MRP4) was found to be extremely high in KU812 cells compared with those of other transporters. Additionally, MRP4 expression levels were found to be relatively high in U937, KG-1 and a blood cell line derived from a healthy subject (RPMI 1788). In addition, to elucidate the contribution of MRP4 to the methotrexate (MTX) distribution in normal blood cells and tissues, [(3)H]MTX was intravenously (i.v.) administered to two groups of rats. Animals in one group received [(3)H]MTX only; the other group was concomitantly administered i.v. MK-571, a typical inhibitor of MRP transporters. No marked difference was observed between the two groups; the Kp values (tissue concentration/plasma concentration) of the concomitant group showed slightly higher values compared with those of the MTX alone group in erythrocytes (1.4 times, P<0.001), spleen (1.3 times, P<0.05) and thymus (1.2 times, P<0.05), respectively. Although in the present study we could not evaluate the direct involvement of MRP4 in blood cancer cells in which MRP4 expression was excessively high, these results suggest a possible functional role of MRP4 in blood cancer cells and albeit only slightly in normal blood cells/tissues.

Previously, we reported that Nardostachys jatamansi (NJ) attenuated cerulein-induced mild acute pancreatitis (AP). In the present study, we investigated the ability of NJ to ameliorate severe acute pancreatitis (SAP) induced by a choline-deficient diet supplemented with ethionine (CDE). An NJ extract was orally administered ad libitum via the water during administration of the CDE. After three days, the CDE was replaced with a normal diet. After four days of normal feeding the mice were sacrificed and the blood and pancreas were obtained for further investigation. NJ treatment reduced SAP-induced pancreatic damage, as shown by histology. NJ treatment also inhibited neutrophil infiltration into the pancreas. NJ also inhibited the secretion of digestive enzymes and cytokine production, and inhibited the activation of mitogen-activated protein kinases (MAPKs) in the SAP-challenged pancreas. These data suggest that NJ protects against pancreatic injury in CDE-induced SAP by deactivating MAPKs.