Tumor resistance to chemotherapy is the major obstacle to employ cisplatin, one of the broadly used chemotherapeutic drugs, for effective treatment of various tumors in the clinic. Most acknowledged mechanisms of cancer resistance to cisplatin focus on increased nuclear DNA repair or detoxicity of cisplatin. We previously demonstrated that there was a unique metabolic profile in cisplatinresistant (CP-r) human epidermoid adenocarcinoma KB-CP 20 and hepatoma BEL 7404-CP 20 cancer cells. In this study, we further defined hyperpolarized mitochondrial membrane potentials (Δψm) in CP-r KB-CP 20 and BEL 7404-CP 20 cells compared to the cisplatin-sensitive (CP-s) KB-3-1 and BEL 7404 cells. Based on the mitochondrial dysfunction, mitaplatin was designed with two mitochondrial-targeting moieties [dichloroacetate (DCA) units] to the axial positions of a six-coordinate Pt(IV) center to sensitize cisplatin resistance. It was found that mitaplatin induced more apoptosis in CP-r KB-CP 20 and BEL 7404-CP 20 cells than that of cisplatin, DCA and cisplatin/DCA compared on an equal molar basis. There was more platinum accumulation in mitaplatin-treated CP-r cells due to enhanced transmembrane permeability of lipophilicity, and mitaplatin also showed special targeting to mitochondria. Moreover, in the case of treatment with mitaplatin, the dramatic collapse of Δψm was shown in a dose-dependent manner, which was confirmed by FACS and confocal microscopic measurements. Reduced glucose utilization of CP-r cells was detected with specifically inhibited phosphorylation of pyruvate dehydrogenase (PDH) at Ser-232, Ser-293, and Ser-300 of the E1α subunit when treated with mitaplatin, which was indicated to modulate the abnormal glycolysis of resistant cells. The present study suggested novel mitochondrial mechanism of mitaplatin circumventing cisplatin resistance toward CP-r cells as a carrier across membrane to produce CP-like cytotoxicity and DCA-like mitochondria-dependent apoptosis. Therefore, mitochondria targeting compounds would be more vulnerable and selective to overcome cisplatin resistance due to the unique metabolic properties of CP-r cancer cells.
mitaplatin; cisplatin; cancer resistance; mitochondrial dysfunction
The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828-1833, 2004; Ma et al., PNAS, 105: 16537-42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ∼83% sequence identity to the transmembrane (TM) domain of the nAChR α1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T2 dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites.
Increased epithelial cell apoptosis in response to lung injury has been implicated in the development of idiopathic pulmonary fibrosis (IPF), but the molecular pathways promoting epithelial cell apoptosis in this disease have yet to be fully identified. Lysophosphatidic acid (LPA), which we have previously demonstrated to mediate bleomycin lung injury–induced fibroblast recruitment and vascular leak in mice and fibroblast recruitment in patients with IPF, is an important regulator of survival and apoptosis in many cell types. We now show that LPA signaling through its receptor LPA1 promotes epithelial cell apoptosis induced by bleomycin injury. The number of apoptotic cells present in the alveolar and bronchial epithelia of LPA1–deficient mice was significantly reduced compared with wild-type mice at Day 3 after bleomycin challenge, as was lung caspase-3 activity. Consistent with these in vivo results, we found that LPA signaling through LPA1 induced apoptosis in normal human bronchial epithelial cells in culture. LPA-LPA1 signaling appeared to specifically mediate anoikis, the apoptosis of anchorage-dependent cells induced by their detachment. Similarly, LPA negatively regulated attachment of R3/1 rat alveolar epithelial cell line cells. In contrast, LPA signaling through LPA1 promoted the resistance of lung fibroblasts to apoptosis, which has also been implicated in IPF. The ability of LPA-LPA1 signaling to promote epithelial cell apoptosis and fibroblast resistance to apoptosis may therefore contribute to the capacity of this signaling pathway to regulate the development of pulmonary fibrosis after lung injury.
pulmonary fibrosis; apoptosis; epithelial cells, lysophosphatidic acid; LPA1
Mutations in the SFTPC gene, encoding surfactant protein–C (SP-C), are associated with interstitial lung disease (ILD). Knowledge of the intracellular fate of mutant SP-C is essential in the design of therapies to correct trafficking/processing of the proprotein, and to prevent the formation of cytotoxic aggregates. We assessed the potential of a chemical chaperone to correct the trafficking and processing of three disease-associated mutant SP-C proteins. HEK293 cells were stably transfected with wild-type (SP-CWT) or mutant (SP-CL188Q, SP-CΔexon4, or SP-CI73T) SP-C, and cell lines with a similar expression of SP-C mRNA were identified. The effects of the chemical chaperone 4-phenylbutyric acid (PBA) and lysosomotropic drugs on intracellular trafficking to the endolysosomal pathway and the subsequent conversion of SP-C proprotein to mature peptide were assessed. Despite comparable SP-C mRNA expression, proprotein concentrations varied greatly: SP-CI73T was more abundant than SP-CWT and was localized to the cell surface, whereas SP-CΔexon4 was barely detectable. In contrast, SP-CL188Q and SP-CWT proprotein concentrations were comparable, and a small amount of SP-CL188Q was localized to the endolysosomal pathway. PBA treatment restored the trafficking and processing of SP-CL188Q to SP-CWT concentrations, but did not correct the mistrafficking of SP-CI73T or rescue SP-CΔexon4. PBA treatment also promoted the aggregation of SP-C proproteins, including SP-CL188Q. This study provides proof of the principle that a chemical chaperone can correct the mistrafficking and processing of a disease-associated mutant SP-C proprotein.
pulmonary surfactant protein–C; interstitial lung disease; 4-phenylbutyric acid; chemical chaperone
The filamentous fungus genus Rhizopus has traditionally been used for brewing alcoholic beverages and fermented foods in China. The 45,666,236-bp draft genome sequence of R. chinensis CCTCCM201021, isolated from the leaven Daqu, was determined, annotated, and analyzed. Analysis of the sequence might provide insight into the properties of this fungus and lead to its further development for industrial applications.
As a proinflammatory cytokine, interleukin-17 (IL-17) contributes to the inflammation of many autoimmune diseases. We examined IL-17 levels in serum and tissues from patients with chronic hepatitis B virus infection (HBV), and especially evaluated the role of IL-17 in the pathogenesis and progression of liver fibrosis.
Materials and methods
Whole venous blood was obtained from four patient groups: chronic hepatitis B (CHB, n = 47), liver cirrhosis (LC, n = 49), primary hepatocellular carcinoma (PHC, n = 44), chronic liver failure (CLF, n = 33), and a normal control group (n = 20). HBsAg was positive in all patients. Liver biopsy samples were acquired from asymptomatic HBsAg carriers (ASC, n = 35), CHB (n = 57), and LC (n = 31) patients. We performed ELISA to measure IL-17 levels in serum samples, and used reverse RT-PCR to measure IL-17 mRNA levels in peripheral blood mononuclear cells (PBMC). IL-17 protein expression was detected in liver biopsy tissues by immunohistochemistry.
Compared to normal controls, serum IL-17 protein and mRNA levels were significantly higher in the four infection groups. LC patients exhibited the highest serum IL-17 and PBMC mRNA levels. No significant differences were found between the other three groups. High levels of IL-17 were also observed in tissues from CHB and LC patients, compared to ASC. IL-17 expression was mainly located in the portal area and was positively correlated with inflammation grade and fibrosis stage.
IL-17 expression was found to be increased with increasing degrees of liver fibrosis. This suggests that IL-17 may not only induce the inflammation, but also contribute to disease progression and chronicity.
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5306959258322482
Asymptomatic; Hepatitis B surface antigen Carriers; Interleukin-17; Hepatitis B virus; Chronic hepatitis B; Liver cirrhosis; Primary hepatitis carcinoma; Chronic liver failure; Fibrosis
Only in recent years have phospholipase A2 enzymes (PLA2s) emerged as cancer targets. In this work, we report the first detection of elevated PLA2 activities in plasma from patients with colorectal, lung, pancreatic, and bladder cancers as compared to healthy controls. Independent sets of clinical plasma samples were obtained from two different sites. The first set was from patients with colorectal cancer (CRC; n = 38) and healthy controls (n = 77). The second set was from patients with lung (n = 95), bladder (n = 31), or pancreatic cancers (n = 38), and healthy controls (n = 79). PLA2 activities were analyzed by a validated quantitative fluorescent assay method and subtype PLA2 activities were defined in the presence of selective inhibitors. The natural PLA2 activity, as well as each subtype of PLA2 activity was elevated in each cancer group as compared to healthy controls. PLA2 activities were increased in late stage vs. early stage cases in CRC. PLA2 activities were not influenced by sex, smoking, alcohol consumption, or body-mass index (BMI). Samples from the two independent sites confirmed the results. Plasma PLA2 activities had approximately 70% specificity and sensitivity to detect cancer. The marker and targeting values of PLA2 activity have been suggested.
To investigate the expressions of type I collagen, α2 integrin and β1 integrin in the posterior sclera of guinea pigs with defocus myopia and whether basic fibroblast growth factor (bFGF) injection inhibits the formation and development of myopia by upregulating the expression of type I collagen, α2 integrin and β1 integrin.
After 14 days of treatment, the refractive state and axial length were measured and the levels of type I collagen, α2 integrin and β1 integrin were assayed in the posterior sclerae of groups of guinea pigs that wore a monocular -7D polymethylmethacrylate (PMMA) lens or had -7D lens wear followed by the peribulbar injection of Phosphate Buffer Solution (PBS) or bFGF. The untreated fellow eye served as a control. Guinea pigs with no treatment served as normal group.
The results showed that 14 days of monocular defocus increased axial eye length and refraction, while bFGF delivery inhibited them markedly. Further, it was also found that the monocular -7D lens could decrease the levels of type I collagen, α2 integrin and β1 integrin expressions, while, unlike PBS, bFGF increased them significantly in comparison to contralateral control eyes and normal eyes.
bFGF can prevent the formation and development of defocus myopia by upregulating the expressions of type I collagen, α2 integrin and β1 integrin. Taken together, our results demonstrate that bFGF promotes sclera remodeling to prevent myopia in guinea pigs.
defocus myopia; type I collagen; α2 integrin; β1 integrin; bFGF
Bacillus licheniformis CGMCC3963 is an important mao-tai flavor-producing strain. It was isolated from the starter (Daqu) of a Chinese mao-tai-flavor liquor fermentation process with solid-state fermentation. We report its genome of 4,525,096 bp here. Many potential insertion genes that are responsible for the unique properties of B. licheniformis CGMCC3963 in mao-tai-flavor liquor production were identified.
Posttranslational modifications (PTMs) of proteins are responsible for sensing and transducing signals to regulate various cellular functions and signaling events. S-nitrosylation (SNO) is one of the most important and universal PTMs. With the avalanche of protein sequences generated in the post-genomic age, it is highly desired to develop computational methods for timely identifying the exact SNO sites in proteins because this kind of information is very useful for both basic research and drug development. Here, a new predictor, called iSNO-PseAAC, was developed for identifying the SNO sites in proteins by incorporating the position-specific amino acid propensity (PSAAP) into the general form of pseudo amino acid composition (PseAAC). The predictor was implemented using the conditional random field (CRF) algorithm. As a demonstration, a benchmark dataset was constructed that contains 731 SNO sites and 810 non-SNO sites. To reduce the homology bias, none of these sites were derived from the proteins that had pairwise sequence identity to any other. It was observed that the overall cross-validation success rate achieved by iSNO-PseAAC in identifying nitrosylated proteins on an independent dataset was over 90%, indicating that the new predictor is quite promising. Furthermore, a user-friendly web-server for iSNO-PseAAC was established at http://app.aporc.org/iSNO-PseAAC/, by which users can easily obtain the desired results without the need to follow the mathematical equations involved during the process of developing the prediction method. It is anticipated that iSNO-PseAAC may become a useful high throughput tool for identifying the SNO sites, or at the very least play a complementary role to the existing methods in this area.
Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionary conserved energy sensor sensitive to changes in cellular AMP/ATP ratio which is activated by phosphorylation (pAMPK). pAMPK levels decrease in peripheral tissues with age, but whether this also occurs in the aged brain, and how this contributes to the ability of the aged brain to cope with ischemic stress is unknown. This study investigated the activation of AMPK and the response to AMPK inhibition after induced stroke in both young and aged male mice. Baseline levels of phosphorylated AMPK were higher in aged brains compared to young mice. Stroke-induced a robust activation of AMPK in young mice, yet this response was muted in the aged brain. Young mice had larger infarct volumes compared with aged animals; however, more severe behavioral deficits and higher mortality were seen in aged mice after stroke. Inhibition of AMPK with Compound C decreased infarct size in young animals, but had no effect in aged mice. Compound C administration led to a reduction in brain ATP levels and induced hypothermia, which led to enhanced neuroprotection in young but not aged mice. This work demonstrates that aging increases baseline brain pAMPK levels; aged mice have a muted stroke-induced pAMPK response; and that AMPK inhibition and hypothermia are less efficacious neuroprotective agents in the aged brain. This has important translational relevance for the development of neuroprotective agents in preclinical models and our understanding of the enhanced metabolic stress experienced by the aged brain.
Aging; AMPK; Compound C; Hypothermia; Ischemic stroke; Middle cerebral artery occlusion (MCAO)
β-Lactamases are important antibiotic resistance determinants expressed by bacteria. By studying the mechanistic properties of β-lactamases, we can identify opportunities to circumvent resistance through the design of novel inhibitors. Comparative amino acid sequence analysis of class A β-lactamases reveals that many enzymes possess a localized positively charged residue (e.g., R220, R244, or R276) that is critical for interactions with β-lactams and β-lactamase inhibitors. To better understand the contribution of these residues to the catalytic process, we explored the roles of R220 and E276 in KPC-2, a class A β-lactamase that inactivates carbapenems and β-lactamase inhibitors. Our study reveals that substitutions at R220 of KPC-2 selectively impact catalytic activity toward substrates (50% or greater reduction in kcat/Km). In addition, we find that residue 220 is central to the mechanism of β-lactamase inhibition/inactivation. Among the variants tested at Ambler position 220, the R220K enzyme is relatively “inhibitor susceptible” (Ki of 14 ± 1 μM for clavulanic acid versus Ki of 25 ± 2 μM for KPC-2). Specifically, the R220K enzyme is impaired in its ability to hydrolyze clavulanic acid compared to KPC-2. In contrast, the R220M substitution enzyme demonstrates increased Km values for β-lactamase inhibitors (>100 μM for clavulanic acid versus 25 ± 3 μM for the wild type [WT]), which results in inhibitor resistance. Unlike other class A β-lactamases (i.e., SHV-1 and TEM-1), the amino acid present at residue 276 plays a structural rather than kinetic role with substrates or inhibitors. To rationalize these findings, we constructed molecular models of clavulanic acid docked into the active sites of KPC-2 and the “relatively” clavulanic acid-susceptible R220K variant. These models suggest that a major 3.5-Å shift occurs of residue E276 in the R220K variant toward the active S70 site. We anticipate that this shift alters the shape of the active site and the positions of two key water molecules. Modeling also suggests that residue 276 may assist with the positioning of the substrate and inhibitor in the active site. These biochemical and molecular modeling insights bring us one step closer to understanding important structure-activity relationships that define the catalytic and inhibitor-resistant profile of KPC-2 and can assist the design of novel compounds.
Demyelination occurs widely in neurodegenerative diseases. Progesterone has neuroprotective effects, is known to reduce the clinical scores and the inflammatory response. Progesterone also promotes remyelination in experimental autoimmune encephalomyelitis and cuprizone-induced demyelinating brain. However, it still remains unclear whether progesterone can alleviate neural behavioral deficits and demyelination with degeneration of oligodendroglial cells in cuprizone-induced mice. In this study, mice were fed with 0.2% cuprizone to induce demyelination, and treated with progesterone to test its potential protective effect on neural behavioral deficits, demyelination and degeneration of oligodendroglial cells. Our results showed noticeable alleviation of neural behavioral deficits following progesterone treatment as assessed by changes in average body weight, and activity during the open field and Rota-rod tests when compared with the vehicle treated cuprizone group. Progesterone treatment alleviated demyelination as shown by Luxol fast blue staining, MBP immunohistochemical staining, and electron microscopy. There was an obvious decrease in TUNEL and Caspase-3-positive apoptotic cells, and an increase in the number of oligodendroglial cells staining positive for PDGFRα, Olig2, Sox10 and CC-1 antibody in the brains of cuprizone-induced mice after progesterone administration. These results indicate that progesterone can alleviate neural behavioral deficits and demyelination against oligodendroglial cell degeneration in cuprizone-induced mice.
We present an ultrasound (US)-guided diffuse optical tomography for mapping tumor deoxyhemoglobin (deoxyHb) and oxyhemoglobin (oxyHb) concentrations in blood phantoms and in in-vivo patients. Because oxyHb and deoxyHb respond differently at different wavelengths, four laser diodes of wavelengths 740 nm, 780 nm, 808 nm and 830 nm were used in the study. Tumor model experiments were performed using phantoms of different hemoglobin oxygen saturations (14%-89%) representing hemoglobin oxygenation in tissue. Targets of different sizes and located at different depths were used to validate the accuracy of oxygen saturation estimation. The absolute deviations between the estimated hemoglobin oxygen saturations obtained from reconstructed absorption maps and oxygen measurements obtained using a pO2 electrode were less than 8% over the measured range of oxygen saturation. An inhomogeneous concentric blood phantom of deoxygenated center core and oxygenated outer shell was imaged and deoxyHb and oxyHb maps revealed corresponding distributions which correlated well with inhomogeneous deoxy- and oxy-distributions frequently seen in breast cancers. Clinical examples are given to demonstrate the utility of US-guided optical tomography in mapping heterogeneous deoxyHb and oxyHb distributions in breast cancers.
Diffuse optical tomography; Ultrasound; Breast cancer; Tumor hypoxia; Tumor oxygen saturation
A new method for the construction of 2-substituted and 2,2-disubstituted chromans via Pd-catalyzed carboetherification reactions between aryl/alkeny halides and 2-(but-3-en-1-yl) phenols is described. These reactions effect formation of a C–O bond and a C–C bond to afford the chroman products in good yield, and also provide stereoselective access to tricyclic chroman derivatives.
Candida parapsilosis CCTCC M203011 catalyzes the stereoinversion of (R)-1-phenyl-1,2-ethanediol (PED) through oxidation and reduction. Its NAD+-linked (R)-carbonyl reductase (RCR) catalyzes the oxidization of (R)-PED to 2-hydroxyacetophenone (HAP), and its NADPH-dependent (S)-carbonyl reductase (SCR) catalyzes the reduction of HAP to (S)-PED. The reactions require NAD+ and NADPH as cofactors. However, even if NAD+ and NADPH are added, the biotransformation of (S)-PED from the (R)-enantiomer by an Escherichia coli strain co-expressing RCR and SCR is slow and gives low yields, probably as a result of insufficient or imbalanced redox cofactors. To prepare (S)-PED from the (R)-enantiomer in one-step efficiently, plus redox cofactor regeneration, we introduced pyridine nucleotide transhydrogenases (PNTs) from E. coli to the metabolic pathway of (S)-PED.
The PNTs were successfully introduced into the E. coli strain RSAB. Most of the PNT activities occurred in the cell membrane of E. coli. The introduction of PNTs increased intracellular NAD+ and NADH concentrations and decreased the NADPH pool without affecting the total nucleotide concentration and cell growth properties. The presence of PNTs increased the NADH/NAD+ ratio slightly and reduced the NADPH/NADP+ ratio about two-fold; the ratio of NADPH/NADP+ to NADH/NAD+ was reduced from 36 to 17. So, the PNTs rebalanced the cofactor pathways: the rate of RCR was increased, while the rate of SCR was decreased. When the ratio of NAD+/NADPH was 3.0 or higher, the RSAB strain produced (S)-PED with the highest optical purity, 97.4%, and a yield of 95.2% at 6 h. The introduction of PNTs stimulated increases of 51.5% and 80.6%, respectively, in optical purity and yield, and simultaneously reduced the reaction time seven-fold.
In this work, PNTs were introduced into E. coli to rebalance the cofactor pools within the engineered (S)-PED pathways. The efficient one-step production of (S)-PED plus NAD+–NADPH in-situ regeneration was realized. This work provided new insights into cofactor rebalancing pathways, using metabolic engineering methods, for efficient chiral alcohol production.
Redox cofactor regeneration; Chiral alcohol; Alcohol dehydrogenases/reductases; Metabolic pathway; One-step stereoinversion
In a 90-day study, Sprague Dawley rats were fed transgenic T1C-1 rice expressing Cry1C protein and were compared with rats fed non-transgenic parental rice Minghui 63 and rats fed a basal diet. No adverse effects on animal behavior or weight gain were observed during the study. Blood samples were collected and analyzed, and standard hematological and biochemical parameters were compared. A few of these parameters were found to be significantly different, but were within the normal reference intervals for rats of this breed and age, and were thus not considered to be treatment-related. Following sacrifice, a large number of organs were weighed, and macroscopic and histopathological examinations were performed with no changes reported. The aim of this study was to use a known animal model to determine the safety of the genetically modified (GM) rice T1C-1. The results showed no adverse or toxic effects due to T1C-1 rice when tested in this 90-day study.
This study was designed to determine the therapeutic efficacy and safety of the Shi-cha capsule, a Chinese herbal formula, in the treatment of patients with wind-cold type common cold. In our multi-center, prospective, double-blind, randomized, placebo-controlled, dose-escalation trial, patients with wind-cold type common cold received 0.6 g of Shi-cha capsule plus 0.6 g placebo (group A), 1.2 g of Shi-cha capsule (group B), or 1.2 g placebo (group C), three times daily for 3 days and followed up to 10 days. The primary end point was all symptom duration. The secondary end points were main symptom duration, minor symptom duration, the changes in cumulative symptom score, main symptom score, and minor symptom score 4 days after the treatment, as well as adverse events. A total of 377 patients were recruited and 360 met the inclusive criteria; 120 patients constituted each treatment group. Compared with patients in group C, patients in groups A and B had significant improvement in the all symptom duration, main symptom duration, minor symptom duration, as well as change from baseline of cumulative symptom score, main symptom score, and minor symptom score at day 4. The symptom durations and scores showed slight superiority of group B over group A, although these differences were not statistically significant. There were no differences in adverse events. The Shi-cha capsule is efficacious and safe for the treatment of patients with wind-cold type common cold. Larger trials are required to fully assess the benefits and safety of this treatment for common cold.
Systemic and intrathecal administration of derivatives of a nonpsychoactive component of marijuana significantly suppresses chronic inflammatory and neuropathic pain, without causing analgesic tolerance, in several rodent models.
Certain types of nonpsychoactive cannabinoids can potentiate glycine receptors (GlyRs), an important target for nociceptive regulation at the spinal level. However, little is known about the potential and mechanism of glycinergic cannabinoids for chronic pain treatment. We report that systemic and intrathecal administration of cannabidiol (CBD), a major nonpsychoactive component of marijuana, and its modified derivatives significantly suppress chronic inflammatory and neuropathic pain without causing apparent analgesic tolerance in rodents. The cannabinoids significantly potentiate glycine currents in dorsal horn neurons in rat spinal cord slices. The analgesic potency of 11 structurally similar cannabinoids is positively correlated with cannabinoid potentiation of the α3 GlyRs. In contrast, the cannabinoid analgesia is neither correlated with their binding affinity for CB1 and CB2 receptors nor with their psychoactive side effects. NMR analysis reveals a direct interaction between CBD and S296 in the third transmembrane domain of purified α3 GlyR. The cannabinoid-induced analgesic effect is absent in mice lacking the α3 GlyRs. Our findings suggest that the α3 GlyRs mediate glycinergic cannabinoid-induced suppression of chronic pain. These cannabinoids may represent a novel class of therapeutic agents for the treatment of chronic pain and other diseases involving GlyR dysfunction.
MDR (multidrug-resistance) represents a major obstacle to successful cancer chemotherapy and is usually accomplished by overexpression of P-gp (P-glycoprotein). Much effort has been devoted to developing P-gp inhibitors to modulate MDR. However, none of the inhibitors on the market have been successful. 1416 [1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxyphenylethylamino)propane hydrochloride (phenoprolamine hydrochloride)] is a new VER (verapamil) analogue with a higher IC50 for blocking calcium channel currents than VER. In the present paper, we examined the inhibition effect of 1416 on P-gp both in vitro and in vivo. 1416 significantly enhanced cytotoxicity of VBL (vinblastine) in P-gp-overexpressed human multidrug-resistant K562/ADM (adriamycin) and KBV cells, but had no such effect on the parent K562 and KB cells. The MDR-modulating function of 1416 was further confirmed by increasing intracellular Rh123 (rhodanmine123) content in MDR cells. Human K562/ADM xenograft-nude mice model verified that 1416 potentiates the antitumour activity of VBL in vivo. RT-PCR (reverse transcriptase-PCR) and FACS analysis demonstrated that the expression of MDR1/P-gp was not affected by 1416 treatment. All these observations suggest that 1416 could be a promising agent for overcoming MDR in cancer chemotherapy.
calcium antagonism; multidrug-resistance; P-glycoprotein; phenoprolamine hydrochloride; verapamil; 1416,1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxyphenylethylamino) propane hydrochloride; ADM, adriamycin; CLSM, confocal laser scanning microscopy; IR, inhibitory rate; MDR, multidrug-resistance; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NS, normal saline; P-gp, P-glycoprotein; RF, resistance fold; Rh123, Rhodamine123; RT-PCR, reverse transcriptase-PCR; RTV, relative tumour volumes; TMs, transmembrane helices; VBL, vinblastine; VER, verapamil
The present study was undertaken to determine the reproductive hazards of Di-(2-ethylhexyl)-phthalate (DEHP) on mouse spermatozoa and embryos in vitro and genomic changes in vivo. Direct low-level DEHP exposure (1 μg/ml) on spermatozoa and embryos was investigated by in vitro fertilization (IVF) process, culture of preimplanted embryos in DEHP-supplemented medium and embryo transfer to achieve full term development. Big Blue® transgenic mouse model was employed to evaluate the mutagenesis of testicular genome with in vivo exposure concentration of DEHP (500 mg/kg/day). Generally, DEHP-treated spermatozoa (1 μg/ml, 30 min) presented reduced fertilization ability (P<0.05) and the resultant embryos had decreased developmental potential compared to DMSO controls (P<0.05). Meanwhile, the transferred 2-cell stage embryos derived from treated spermatozoa also exhibited decreased birth rate than that of control (P<0.05). When fertilized oocytes or 2-cell stage embryos were recovered by in vivo fertilization (without treatment) and then exposed to DEHP, the subsequent development proceed to blastocysts was different, fertilized oocytes were significantly affected (P<0.05) whereas developmental progression of 2-cell stage embryos was similar to controls (P>0.05). Testes of the Big Blue® transgenic mice treated with DEHP for 4 weeks indicated an approximately 3-fold increase in genomic DNA mutation frequency compared with controls (P<0.05). These findings unveiled the hazardous effects of direct low-level exposure of DEHP on spermatozoa's fertilization ability as well as embryonic development, and proved that in vivo DEHP exposure posed mutagenic risks in the reproductive organ – at least in testes, are of great concern to human male reproductive health.
Extracellular guidance cues steer axons towards their targets by eliciting morphological changes in the growth cone. A key part of this process is the asymmetric recruitment of the cytoplasmic scaffolding protein MIG-10 (lamellipodin). MIG-10 is thought to asymmetrically promote outgrowth by inducing actin polymerization. However, the mechanism that links MIG-10 to actin polymerization is not known. We have identified the actin regulatory protein ABI-1 as a partner for MIG-10 that can mediate its outgrowth-promoting activity. The SH3 domain of ABI-1 binds to MIG-10, and loss of function of either of these proteins causes similar axon guidance defects. Like MIG-10, ABI-1 functions in both the attractive UNC-6 (netrin) pathway and the repulsive SLT-1 (slit) pathway. Dosage sensitive genetic interactions indicate that MIG-10 functions with ABI-1 and WVE-1 to mediate axon guidance. Epistasis analysis reveals that ABI-1 and WVE-1 function downstream of MIG-10 to mediate its outgrowth-promoting activity. Moreover, experiments with cultured mammalian cells suggest that the interaction between MIG-10 and ABI-1 mediates a conserved mechanism that promotes formation of lamellipodia. Together, these observations suggest that MIG-10 interacts with ABI-1 and WVE-1 to mediate the UNC-6 and SLT-1 guidance pathways.
To form neural circuits, axons must navigate through the developing nervous system to reach their correct targets. Axon navigation is led by the growth cone, a structure at the tip of the growing axon that responds to extracellular guidance cues. Many of these guidance cues and their receptors have been identified. However, much less is known about the internal signaling events that give rise to the structural changes required for growth cone steering. A key component of the internal response is MIG-10, a protein that becomes asymmetrically localized in response to the extracellular cues. MIG-10 is thought to serve as a scaffold that can spatially control outgrowth-promoting proteins within the growth cone. However, we do not know the identity of the outgrowth-promoting proteins that associate with MIG-10. Here we report that MIG-10 associates physically with the actin regulatory protein ABI-1. We present genetic evidence indicating that ABI-1 functions downstream of MIG-10 to mediate its outgrowth-promoting activity. Additional genetic evidence indicates that these proteins function in both attractive and repulsive guidance signaling pathways. We also present evidence suggesting that the connection between MIG-10 and ABI-1 represents a phylogenetically conserved mechanism for the control of cellular outgrowth.
plays an important role in immunoregulation; it induces the differentiation of Th1 together with the homeobox transcription factor gene Hlx. Recent studies show that T-bet and Th1-associated factors are critical in regulating tumor development. However, the contributions of Hlx in the occurrence and development of cancer remain unknown. In this study, the Hlx, T-bet, Runx3, and IFN-γ were measured in PBMC from patients with gastric cancer and the correlation between Hlx and T-bet or IFN-γ was assessed. The expression levels of Hlx, T-bet, and IFN-γwere significantly decreased, and there was a positive correlation between Hlx and T-bet or IFN-γ. In addition, the Runx3 expression was also downregulated with the lower T-bet mRNA level. These results suggested that the decreased Hlx expression was closely associated with T-bet and Runx3 downregulations and may contribute to the development of gastric cancer.
Mucinous adenocarcinoma of the lung is a subtype of highly invasive pulmonary tumors and is associated with decreased or absent expression of the transcription factor NK2 homeobox 1 (NKX2-1; also known as TTF-1). Here, we show that haploinsufficiency of Nkx2-1 in combination with oncogenic KrasG12D, but not with oncogenic EGFRL858R, caused pulmonary tumors in transgenic mice that were phenotypically similar to human mucinous adenocarcinomas. Gene expression patterns distinguished tumor goblet (mucous) cells from nontumorigenic airway and intestinal goblet cells. Expression of NKX2-1 inhibited urethane and oncogenic KrasG12D-induced tumorigenesis in vivo. Haploinsufficiency of Nkx2-1 enhanced KrasG12D-mediated tumor progression, but reduced EGFRL858R-mediated progression. Genome-wide analysis of gene expression demonstrated that a set of genes induced in mucinous tumors was shared with genes induced in a nontumorigenic chronic lung disease, while a distinct subset of genes was specific to mucinous tumors. ChIP with massively parallel DNA sequencing identified a direct association of NKX2-1 with the genes induced in mucinous tumors. NKX2-1 associated with the AP-1 binding element as well as the canonical NKX2-1 binding element. NKX2-1 inhibited both AP-1 activity and tumor colony formation in vitro. These data demonstrate that NKX2-1 functions in a context-dependent manner in lung tumorigenesis and inhibits KrasG12D-driven mucinous pulmonary adenocarcinoma.