Stimulation of oncogenic metabotropic glutamate receptor 1 in melanoma cells activates ERK1/2 via PKCepsilon

Metabotropic glutamate receptor 1 (Grm1, formerly mGluR1) is a G protein coupled receptor (GPCR) normally expressed and functional in the central nervous system. Studies of our transgenic mouse melanoma model (TG-3) revealed that ectopic expression of Grm1 in melanocytes is sufficient to induce melanoma development in vivo [P.M. Pollock, K. Cohen-Solal, R. Sood, J. Namkoong, J.J. Martino, A. Koganti, H. Zhu, C. Robbins, I. Makalowska, S.S. Shin, Y. Marin, K.G. Roberts, L.M. Yudt, A. Chen, J. Cheng, A. Incao, H.W. Pinkett, C.L. Graham, K. Dunn, S.M. Crespo-Carbone, K.R. Mackason, K.B. Ryan, D. Sinsimer, J. Goydos, K.R. Reuhl, M. Eckhaus, P.S. Meltzer, W.J. Pavan, J.M. Trent, S. Chen, Nat. Genet. 34 (2003) 108-112.]. We have established and characterized several cell lines in vitro from independent mouse melanoma tumors [Y.E. Marín, J. Namkoong, S.S. Shin, J. Raines, K. Degenhardt, E. White, S. Chen, Neuropharmacol. 49 (2005) 70-79.]. These cell lines are useful tools in the studies of signaling events that may be mediated by Grm1 in transformed melanocytes. Here we show that stimulation of Grm1 by l-quisqualate, a group I metabotropic glutamate receptor agonist, results in inositol triphosphate (IP3) accumulation, and the activation of ERK1/2 in these cell lines. IP3 accumulation and ERK1/2 activation were inhibited by pretreatment of the tumor cells with a Grm1-specific antagonist (LY367385) or by dominant negative mutants of Grm1, demonstrating the specificity of these events. We also show that ERK1/2 activation by Grm1 was PKC-dependent, but cAMP and PKA-independent. PKCepsilon was shown to play a pivotal role in Grm1-mediated ERK1/2 phosphorylation. Insights into the signaling cascades mediated by Grm1 in melanoma cells may aid in the identification of key molecular targets for the future design of combined therapies for melanoma.     

Reduction of chronic graft injury with a new HTK-based preservation solution in a murine heart transplantation model

Objective

Aim of this study was to evaluate a new histidine-tryptophan-ketoglutarate (HTK)-based preservation solution on chronic isograft injury in comparison to traditional HTK solution.

Methods

Hearts of C57BL/6J (H-2b) mice were stored for 15 h in 0–4 °C cold preservation solution and then transplanted heterotopically into C57BL/6J (H-2b) mice. Three groups were evaluated: HTK, the base solution of a new preservation solution and hearts without cold ischemia (control). Time to restoration of heartbeat was measured (re-beating time). Strength of the heartbeat was palpated daily and scored on a 4-level scale (palpation score). Animals were sacrificed after 60 days of observation (24 h for TGF-β expression). The transplanted hearts were evaluated histologically for myocardial damage, vasculopathy and interstitial fibrosis. TGF-β expression was assessed immunohistologically. All investigators were blinded to the groups. ANOVA and LSD post hoc test were used for statistical analysis.

Results

The re-beating time was significantly shorter in hearts stored in the new solution (10.3 ± 2.6 min vs. HTK 14.2 ± 4.1 min; p < 0.05). The palpation score was significantly higher in hearts stored in the new solution (2.3 ± 0.4 vs. HTK 1.6 ± 0.5; p < 0.01). Hearts stored in the new solution showed a lower myocardial injury score (1.8 ± 0.2 vs. HTK 2.2 ± 0.7), less interstitial fibrosis (4.8 ± 1.9% vs. HTK 8.5 ± 3.8%, p < 0.05), less vasculopathy (14.7 ± 6.9% vs. 22.0 ± 23.2%; p = 0.06) and lower TGF-β1-expression (6.6 ± 1.4% vs. HTK 12.0 ± 4.6%).

Conclusion

The new HTK-based solution reduces the chronic isograft injury. This protective effect is likely achieved through several modifications and supplements into the new solution like N-acetyl-l-histidine, glycine, alanine, arginine and sucrose.     

The influence of cholesterol and lipid metabolism on host cell structure and hepatitis C virus replication.

The hepatitis C virus (HCV) replicates on a membrane protein complex composed of viral proteins, replicating RNA, and altered cellular membranes. Small-molecule inhibitors of cellular lipid-cholesterol metabolism such as 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 all show a negative effect on HCV replication. Perturbation of host cell lipid and cholesterol metabolism can disrupt replication complexes by altering membranous structures where replication occurs. Changes in cholesterol and (or) lipid composition can have a general effect on membrane structure. Alternatively, metabolic changes can exert a more subtle influence over replication complexes by altering localization of host proteins through alterations in lipid anchoring. Here, we use Huh-7 cells harboring subgenomic HCV replicons to demonstrate that 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 do not disrupt the membranous web where replication occurs, whereas cholesterol-depleting agents such as beta-cyclodextrin do. Cellular imaging suggests that the HCV RNA can remain associated with subcellular compartments connected with replication complexes in the presence of metabolic inhibitors. Therefore, at least 2 different molecular mechanisms are possible for the inhibition of HCV replication through the modulation of cellular lipid and cholesterol metabolism.     

Cloning and characterization of a novel human RNA binding protein gene PNO1.

Present work reported the cloning and characterization of a human novel RNA binding gene Partner of NOB1 (PNO1), with a length of 1637bp and a putative open reading frame of 759 bp, isolated from human kidney. It is composed of seven exons and is localized on chromosome 2p14. Western blot showed that the molecular weight of PNO1 is about 35kDa. RT-PCR results in 16 human tissues indicated that PNO1 is expressed mainly in liver, lung, spleen and kidney, slightly in thymus, testis, ovary, respectively, but not in heart, brain, skeletal muscle, placenta, pancreas, prostate, small intestine, colon and peripheral blood leukocytes. GFP fusion expression in mammalian cells exhibited its localization in the nucleus, especially in nucleoli. Subcellular localization of thirteen GFP fusion PNO1 deletion proteins showed that the region of 92-230 aa is solely responsible for its nucleolar retention, and KH domain alone is not sufficient for nucleolar retention. The PNO1 family shows significant conservation in both eukaryotes and prokaryotes.     

Changes in Glial Cell Line-derived Neurotrophic Factor Expression in the Rostral and Caudal Stumps of the Transected Adult Rat Spinal Cord

Limited information is available regarding the role of endogenous Glial cell line-derived neurotrophic factor (GDNF) in the spinal cord following transection injury. The present study investigated the possible role of GDNF in injured spinal cords following transection injury (T₉–T₁₀) in adult rats. The locomotor function recovery of animals by the BBB (Basso, Beattie, Bresnahan) scale score showed that hindlimb support and stepping function increased gradually from 7 days post operation (dpo) to 21 dpo. However, the locomotion function in the hindlimbs decreased effectively in GDNF-antibody treated rats. GDNF immunoreactivty in neurons in the ventral horn of the rostral stump was stained strongly at 3 and 7 dpo, and in the caudal stump at 14 dpo, while immunostaining in astrocytes was also seen at all time-points after transection injury. Western blot showed that the level of GDNF protein underwent a rapid decrease at 7 dpo in both stumps, and was followed by a partial recovery at a later time-point, when compared with the sham-operated group. GDNF mRNA-positive signals were detected in neurons of the ventral horn, especially in lamina IX. No regenerative fibers from corticospinal tract can be seen in the caudal segment near the injury site using BDA tracing technique. No somatosensory evoked potentials (SEP) could be recorded throughout the experimental period as well. These findings suggested that intrinsic GDNF in the spinal cord could play an essential role in neuroplasticity. The mechanism may be that GDNF is involved in the regulation of local circuitry in transected spinal cords of adult rats.     

Phylogenetic Analysis of AA-genome Oryza Species (Poaceae) Based on Chloroplast, Mitochondrial, and Nuclear DNA Sequences

Species in the genus Oryza (Poaceae) contain 10 genomic types and are distributed in pan-tropics of the world. To explore phylogenetic relationships of Oryza species having the AA-genome, DNA sequences of the chloroplast trnL intron and trnL-trnF spacer, mitochondrial nad1 intron 2, and nuclear internal transcribed spacer were analyzed, based on materials from 6 cultivated (O. sativa and O. glaberrima) and 13 wild accessions, in addition to a CC-genome species (O. officinalis) that was used as an outgroup. Analyses of the combined sequence data set from different sources provide a much better resolution of the AA-genome species than the individual data set, indicating the limitation of a single gene in phylogenetic reconstruction. The phylogeny based on the combined data set demonstrated an apparent grouping of the AA-genome Oryza species that was well associated with their geographic origin, although the Australian O. meridionalis showed its affinity with the African species. The geographic pattern of the phylogenetic relationship was probably attributed to the frequent genetic exchange and introgression among the AA-genome species from the same continents. In addition, Asian cultivated rice O. sativa showed its close relation to O. rufipogon and O. nivara, whereas African cultivated rice O. glaberrima was closely linked to O. barthii and O. longistaminata, indicating the independent domestication of the two cultivated species in different geographic locations.     

Antimicrobial treatment of grapes using sodium hypochlorite in winemaking and its effects on the chemical and sensory characteristics of wines

This study was performed to examine the use of NaOCl as an alternative antimicrobial compound in winemaking because of the potential health problems that may arise as a result of the use of SO2. For this, the blank (non-treated), control (SO2-added), and sample (NaOCl-treated) wines were made, and microbial and chemical changes including sensory characteristics were analyzed during the fermentation periods. Treatment of grapes with NaOCl decreased the initial contaminating microbial population in grape must, resulting in higher growth of yeast and lactic acid bacteria. After 200 days of fermentation, the chemical analysis of sample wine revealed that it had higher ethanol content, redness (a*), and concentrations of fruity ester compounds and lower total acidity than the control. In the sensory analyses, the sample wine obtained a higher overall acceptability score (5.70) than the control (4.26). This result reveals that NaOCl can be used as an alternative to SO2 in winemaking for inhibiting the growth of contaminating microorganisms.     

Association of the CTLA4 Gene with Graves' Disease in the Chinese Han Population

To determine whether genetic heterogeneity exists in patients with Graves'' disease (GD), the cytotoxic T-lymphocyte associated 4 (CTLA-4) gene, which is implicated a susceptibility gene for GD by considerable genetic and immunological evidence, was used for association analysis in a Chinese Han cohort recruited from various geographic regions. Our association study for the SNPs in the CTLA4 gene in 2640 GD patients and 2204 control subjects confirmed that CTLA4 is the susceptibility gene for GD in the Chinese Han population. Moreover, the logistic regression analysis in the combined Chinese Han cohort revealed that SNP rs231779 (allele frequencies p = 2.81×10⁻⁹, OR = 1.35, and genotype distributions p = 2.75×10⁻⁹, OR = 1.42) is likely the susceptibility variant for GD. Interestingly, the logistic regression analysis revealed that SNP rs35219727 may be the susceptibility variant to GD in the Shandong population; however, SNP, rs231779 in the CTLA4 gene probably independently confers GD susceptibility in the Xuzhou and southern China populations. These data suggest that the susceptibility variants of the CTLA4 gene varied between the different geographic populations with GD.     

Exogenous Glycine Betaine Reduces Drought Damage by Mediating Osmotic Adjustment and Enhancing Antioxidant Defense in <i>Phoebe hunanensis</i>

Drought stress negatively impacts growth and physiological processes in plants. The foliar application of glycine betaine (GB) is an effective and low-cost approach to improve the drought tolerance of trees. This study examined the effect of exogenously applied GB on the cell membrane permeability, osmotic adjustment, and antioxidant enzyme activities of Phoebe hunanensis Hand.-Mazz under drought stress. Two levels (0 and 800 mL) of water irrigation were tested under different applied GB concentrations (0, 50, 100, and 200 mM). Drought stress decreased the relative water content by 58.5% while increased the electric conductivity, malondialdehyde, proline, soluble proteins, soluble sugars, and antioxidant enzyme activities (superoxide dismutase, catalase, peroxidase) by up to 62.9%, 42.4%, 87.0%, 19.1%, 60.5%, 68.3%, 71.7%, and 83.8%, respectively, on the 25^th day. The foliar application of GB, especially at 100 mM, increased the relative water content of P. hunanensis leaves under drought stress. The concentration of GB from 50 to 100 mM effectively alleviated the improvement of cell membrane permeability and inhibited the accumulation of membrane lipid peroxidation products. Under drought stress, the concentrations of proline, soluble proteins, and soluble sugars in the leaves of P. hunanensis increased as the applied GB concentration was increased and the water stress time was prolonged. Exogenously applied GB decreased oxidative stress and improved antioxidant enzyme activities as compared with treatments without GB application. Furthermore, the physiological and biochemical indexes of P. hunanensis showed a certain dose effect on exogenous GB concentration. These results suggest that GB helps maintain the drought tolerance of P. hunanensis.