The glutamate agonist homocysteine sulfinic acid stimulates glucose uptake through the calcium-dependent AMPK-p38 MAPK-protein kinase C zeta pathway in skeletal muscle cells

Homocysteine sulfinic acid (HCSA) is a homologue of the amino acid cysteine and a selective metabotropic glutamate receptor (mGluR) agonist. However, the metabolic role of HCSA is poorly understood. In this study, we showed that HCSA and glutamate stimulated glucose uptake in C2C12 mouse myoblast cells and increased AMP-activated protein kinase (AMPK) phosphorylation. RT-PCR and Western blot analysis revealed that C2C12 expresses mGluR5. HCSA transiently increased the intracellular calcium concentration. Although α-methyl-4-carboxyphenylglycine, a metabotropic glutamate receptor antagonist, blocked the action of HCSA in intracellular calcium response and AMPK phosphorylation, 6-cyano-7-nitroquinoxaline-2,3-dione, an AMPA antagonist, did not exhibit such effects. Knockdown of mGluR5 with siRNA blocked HCSA-induced AMPK phosphorylation. Pretreatment of cells with STO-609, a calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, blocked HCSA-induced AMPK phosphorylation, and knockdown of CaMKK blocked HCSA-induced AMPK phosphorylation. In addition, HCSA activated p38 mitogen-activated protein kinase (MAPK). Expression of dominant-negative AMPK suppressed HCSA-mediated phosphorylation of p38 MAPK, and inhibition of AMPK and p38 MAPK blocked HCSA-induced glucose uptake. Phosphorylation of protein kinase C ζ (PKCζ) was also increased by HCSA. Pharmacologic inhibition or knockdown of p38 MAPK blocked HCSA-induced PKCζ phosphorylation, and knockdown of PKCζ suppressed the HCSA-induced increase of cell surface GLUT4. The stimulatory effect of HCSA on cell surface GLUT4 was impaired in FITC-conjugated PKCζ siRNA-transfected cells. Together, the above results suggest that HCSA may have a beneficial role in glucose metabolism in skeletal muscle cells via stimulation of AMPK.     

Wnt5a-dopamine D2 receptor interactions regulate dopamine neuron development via extracellular signal-regulated kinase (ERK) activation

The dopamine D2 receptor (D2R) plays an important role in mesencephalic dopaminergic neuronal development, particularly coupled with extracellular signal-regulated kinase (ERK) activation. Wnt5a protein is known to regulate the development of dopaminergic neurons. We analyzed the effect of Wnt5a on dopaminergic neuron development in mesencephalic primary cultures from wild-type (WT) and D2R knock-out (D2R(-/-)) mice. Treatment with Wnt5a increased the number and neuritic length of dopamine neurons in primary mesencephalic neuronal cultures from WT mice, but not from D2R(-/-) mice. The effect of Wnt5a was completely blocked by treatment with D2R antagonist or inhibitors of MAPK or EGFR. Wnt5a-mediated ERK activation in mesencephalic neuronal cultures was inhibited by treatment of D2R antagonist and EGFR inhibitors in WT mice. However, these regulations were not observed for D2R(-/-) mice. Co-immunoprecipitation and displacement of [(3)H]spiperone from D2R by Wnt5a demonstrated that Wnt5a could bind with D2R. This interaction was confirmed by GST pulldown assays demonstrating that the domain including transmembrane domain 4, second extracellular loop, and transmembrane domain 5 of D2R binds to Wnt5a. These results suggest that the interaction between D2R and Wnt5a has an important role in dopamine neuron development in association with EGFR and the ERK pathway.     

A domain in the herpes simplex virus 1 triplex protein VP23 is essential for closure of capsid shells into icosahedral structures

VP23 is a key component of the triplex structure. The triplex, which is unique to herpesviruses, is a complex of three proteins, two molecules of VP23 which interact with a single molecule of VP19C. This structure is important for shell accretion and stability of the protein coat. Previous studies utilized a random transposition mutagenesis approach to identify functional domains of the triplex proteins. In this study, we expand on those findings to determine the key amino acids of VP23 that are required for triplex formation. Using alanine-scanning mutagenesis, we have made mutations in 79 of 318 residues of the VP23 polypeptide. These mutations were screened for function both in the yeast two-hybrid assay for interaction with VP19C and in a genetic complementation assay for the ability to support the replication of a VP23 null mutant virus. These assays identified a number of amino acids that, when altered, abolish VP23 function. Abrogation of virus assembly by a single-amino-acid change bodes well for future development of small-molecule inhibitors of this process. In addition, a number of mutations which localized to a C-terminal region of VP23 (amino acids 205 to 241) were still able to interact with VP19C but were lethal for virus replication when introduced into the herpes simplex virus 1 (HSV-1) KOS genome. The phenotype of many of these mutant viruses was the accumulation of large open capsid shells. This is the first demonstration of capsid shell accumulation in the presence of a lethal VP23 mutation. These data thus identify a new domain of VP23 that is required for or regulates capsid shell closure during virus assembly.     

A combined approach of classical mutagenesis and rational metabolic engineering improves rapamycin biosynthesis and provides insights into methylmalonyl-CoA precursor supply pathway in <Emphasis Type="Italic">Streptomyces hygroscopicus</Emphasis> ATCC 29253

Rapamycin is a macrocyclic polyketide with immunosuppressive, antifungal, and anticancer activity produced by Streptomyces hygroscopicus ATCC 29253. Rapamycin production by a mutant strain (UV2-2) induced by ultraviolet mutagenesis was improved by approximately 3.2-fold (23.6 mg/l) compared to that of the wild-type strain. The comparative analyses of gene expression and intracellular acyl-CoA pools between wild-type and the UV2-2 strains revealed that the increased production of rapamycin in UV2-2 was due to the prolonged expression of rapamycin biosynthetic genes, but a depletion of intracellular methylmalonyl-CoA limited the rapamycin biosynthesis of the UV2-2 strain. Therefore, three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA were evaluated to identify the effective precursor supply pathway that can support the high production of rapamycin: propionyl-CoA carboxylase (PCC), methylmalonyl-CoA mutase, and methylmalonyl-CoA ligase. Among them, only the PCC pathway along with supplementation of propionate was found to be effective for an increase in intracellular pool of methylmalonyl-CoA and rapamycin titers in UV2-2 strain (42.8 mg/l), indicating that the PCC pathway is a major methylmalonyl-CoA supply pathway in the rapamycin producer. These results demonstrated that the combined approach involving traditional mutagenesis and metabolic engineering could be successfully applied to the diagnosis of yield-limiting factors and the enhanced production of industrially and clinically important polyketide compounds.     

Albumin-induced epithelial-mesenchymal transition and ER stress are regulated through a common ROS-c-Src kinase-mTOR pathway: effect of imatinib mesylate

The epithelial-mesenchymal transition (EMT) and endoplasmic reticulum (ER) stress induced by urinary protein, particularly albumin, play an important role in tubulointerstitial injury. However, signaling pathways regulating both albumin-induced EMT and ER stress are not precisely known. We postulated that reactive oxygen species (ROS), c-Src kinase, and mammalian target of rapamysin (mTOR) would act as upstream signaling molecules. We further examined the effect of imatinib mesylate on these processes. All experiments were performed using HK-2 cells, a human proximal tubular cell line. Protein and mRNA expression were measured by Western blot analysis and real-time PCR, respectively. Exposure of tubular cells to albumin (5 mg/ml) for up to 5 days induced EMT in a time-dependent manner, as shown by conversion to the spindle-like morphology, loss of E-cadherin protein, and upregulation of α-smooth muscle actin mRNA and protein. Albumin also induced ER stress as evidenced by phosphorylation of eukaryotic translation initiation factor-2α and increased expression of GRP78 mRNA and protein. Albumin induced ROS, c-Src kinase, and mTOR as well. Antioxidants, c-Src kinase inhibitor (PP2), and mTOR inhibitor (rapamycin) suppressed the albumin-induced EMT and ER stress. Antioxidants and PP2 inhibited the albumin-induced c-Src kinase and mTOR, respectively. Imatinib suppressed the albumin-induced EMT and ER stress via inhibition of ROS and c-Src kinase. Imatinib also inhibited the albumin-induced mRNA expression of MCP-1, VCAM-1, transforming growth factor (TGF)-β1, and collagen I (α1). In conclusion, the ROS-c-Src kinase-mTOR pathway played a central role in the signaling pathway that linked albumin to EMT and ER stress. Imatinib might be beneficial in attenuating the albumin-induced tubular injury.     

Effect of N-2-mercaptopropionyl glycine on exercise-induced cardiac adaptations

The purpose of this study was to test the hypothesis that exercise-induced cardiac adaptations would be attenuated by the free radical scavenger N-2-mercaptopropionyl glycine (MPG). Male Sprague-Dawley rats were divided into four groups (n = 9-13 per group) for 3-4 wk: sedentary (S), S+MPG (100 mg/kg ip daily), exercised on a treadmill (E) (60 min/day, 5 days/wk, at a speed of 20 m/min up a 6° grade in a 6°C room), or E+MPG given 10 min prior to exercise. Additional rats (n = 55) were used to determine acute exercise effects on myocardial redox state [nonprotein nonglutathione sulfhydryls (NPNGSH)] and PI3K/Akt signaling pathway activation. Compared with S, NPNGSH levels were 48% lower in E (P < 0.05) and unchanged in E+MPG (P > 0.05). MPG also attenuated exercise-induced activation of the signaling proteins Akt and S6. Hearts from the 4-wk groups were weighed, and cardiac function was evaluated using an isolated perfused working heart preparation. Similar increases (P < 0.05) in both exercised groups were observed for heart weight and heart weight-to-body weight ratio. Cardiac function improved in E vs. S, as indicated by greater (P < 0.05) external work performed (cardiac output × systolic pressure) and efficiency of external work (work/Vo(2)). MPG prevented these exercise-induced functional improvements. Skeletal muscle mitochondria content increased to similar levels in E and E+MPG. This study provides evidence that free radicals do not play an essential role in the development of exercise-induced cardiac hypertrophy; however, they appear to be involved in functional cardiac adaptations, which may be mediated through the PI3K/Akt pathway.     

Refinement of protein termini in template-based modeling using conformational space annealing

The rapid increase in the number of experimentally determined protein structures in recent years enables us to obtain more reliable protein tertiary structure models than ever by template-based modeling. However, refinement of template-based models beyond the limit available from the best templates is still needed for understanding protein function in atomic detail. In this work, we develop a new method for protein terminus modeling that can be applied to refinement of models with unreliable terminus structures. The energy function for terminus modeling consists of both physics-based and knowledge-based potential terms with carefully optimized relative weights. Effective sampling of both the framework and terminus is performed using the conformational space annealing technique. This method has been tested on a set of termini derived from a nonredundant structure database and two sets of termini from the CASP8 targets. The performance of the terminus modeling method is significantly improved over our previous method that does not employ terminus refinement. It is also comparable or superior to the best server methods tested in CASP8. The success of the current approach suggests that similar strategy may be applied to other types of refinement problems such as loop modeling or secondary structure rearrangement.     

Aromatic radiofluorination and biological evaluation of 2-aryl-6-[18F]fluorobenzothiazoles as a potential positron emission tomography imaging probe for β-amyloid plaques

To develop agents for radionuclide imaging Aβ plaques in vivo, we prepared three fluorine-substituted analogs of arylbenzothiazole class; compound 2 has a high affinity for Aβ (K(i)=5.5nM) and the specific binding to Aβ in fluorescent staining. In preparation for the synthesis of these arylbenzothiazole analogs in radiolabeled form as an Aβ plaques-specific positron emission tomography (PET) imaging probe, we investigated synthetic route suitable for its labeling with the short-lived PET radionuclide fluorine-18 (t(1/2)=110min) and diaryliodonium tosylate precursors (12, 13a-e and 14). 2-Aryl-6-[(18)F]fluorobenzothiazoles ([(18)F]1-3) were synthesized in efficiently short reaction times (40-60min) with high radiochemical yields (19-40%), purities (>95%) and specific activities (85-118GBq/μmol). Tissue distribution studies showed that high radioactivity of [(18)F]2 accumulated in the brain with rapid clearance in healthy mice. Radioactive metabolites were analyzed in brain samples of mice and corresponded to 81% of parent remained by 30min after a tail-vein injection. These results suggest that [(18)F]2 is a promising probe for evaluation of Aβ plaques imaging in brain using PET.     

Oleanane-type triterpene saponins from the bark of Aralia elata and their NF-κB inhibition and PPAR activation signal pathway

Two new oleanane-type triterpene saponins, tarasaponin IV (1) and elatoside L (2), and four known; stipuleanoside R(2) (3), kalopanax-saponin F (4), kalopanax-saponin F methylester (5), and elatoside D (6) were isolated from the bark of Aralia elata. Kalopanax-saponin F methyl ester was isolated from nature for the first time. Their chemical structures were elucidated using the chemical and physical methods as well as good agreement with those of reported in the literature. Oleanane-type triterpene saponins are the main component of A. elata. All compounds were investigated the anti-inflammatory activity. We measured their inhibition of NF-κB and activation of PPARs activities in HepG2 cells using luciferase reporter system. As results, compounds 2 and 4 were found to inhibit NF-κB activation stimulated by TNFα in a dose-dependent manner with IC(50) values of 4.1 and 9.5 μM, respectively, when compared with that of positive control, sulfasalazine (0.9 μM). Compounds 2 and 4 also inhibited TNFα-induced expression of iNOS and COX-2 mRNA. Furthermore, compounds 1-6 were evaluated PPAR activity using PPAR subtype transactivation assays. Among of them, compounds 4-6 significantly increased PPARγ transactivation. However, compounds 4-6 did not activate in any other PPAR subtypes.