Physicochemical Modulation of Agonist-Induced [35S]GTPγS Binding: Implications for Coexistence of Multiple Functional Conformations of Dopamine D1-Like Receptors

Dopamine agonist-stimulated [³⁵S]GTPγS binding to membrane G proteins was studied in select brain regions under experimental conditions that permit the activation of receptor coupling to the G proteins G_i, G_s, or G_q. Agents studied were agonists known to be effective at various dopamine receptor/effector systems and included quinelorane (D₂-like/G_i), SKF38393 (D₁-like/G_q, D₁-like/G_s), SKF85174 (D₁-like/G_s), and SKF83959 (D₁-like/G_q). Dopamine and SKF38393 significantly stimulated [³⁵S]GTPγS binding to normal striatal membranes by 161% and 67% above controls. Deoxycholate, which enhances agonist-induced phospholipase C (PLC) stimulation, markedly enhanced the agonistic effects of dopamine and SKF38393 to 530% and 637% above controls, respectively. The enhancing effects of deoxycholate were reversed if it was washed off the membranes before agonist addition. The thiol-reducing agent, dithiothreitol, completely abolished the effects of SKF38393 and SKF83959, whereas SKF85174 effects were augmented. Agonist responses were concentration-related, and highest efficacies were obtained in the hippocampus, thus paralleling both the brain regional distribution and agonist efficacies previously observed in phosphoinositide hydrolysis assays. These findings suggest that D₁-like receptor conformations that mediate agonist stimulation of G_s/adenylylcyclase may be structurally different from those that mediate G_q/PLC activation. Although the exact mechanism of deoxycholate's effect awaits elucidation, the results are consistent with the emerging concept of functional selectivity whereby deoxycholate could create a membrane environment that facilitates the transformation of the receptor from a conformation that activates G_s/adenylylcyclase to one that favors G_q/PLC signaling.     

Association of insulin receptor and syndecan-1 by insulin with activation of ERK I/II in osteoblast-like UMR-106 cells

Abstract

Insulin plays an important role in various metabolic as well as anabolic actions in cells, including osteoblast cells. In the present study, we explored to determine if insulin receptor could associate with syndecan-1 in response to insulin and such association could lead to the activation of subsequent ERK I/II and alkaline phosphatase (ALP) in osteoblast cells. Insulin rapidly induces the association of insulin receptor with syndecan-1. Synstatin is a specific peptide inhibitor that blocks the binding of syndecan-1 to integrate. In the presence of synstatin, insulin-stimulated ERK I/II activation was dramatically inhibited, suggesting that syndecan-1/integrin interaction is essential in the activation of ERK I/II by insulin. Pretreatment of synstatin also inhibited the insulin-stimulated ALP activity. Taken together, these results suggest that insulin stimulates the association of insulin receptor with syndecan-1 and the complex formation of syndecan-1 and integrin could play an important role in ERK I/II–ALP signaling pathway in osteoblast cells.     

Genoprotection and genotoxicity of green tea (Camellia sinensis): Are they two sides of the same redox coin?

Abstract

Objectives

Regular intake of green tea associates with lower DNA damage and increased resistance of DNA to oxidant challenge. However, in vitro pro-oxidant effects of green tea have been reported. Both effects could be mediated by hydrogen peroxide (H₂O₂) which is generated by autoxidation of tea catechins. In large amounts, H₂O₂ is genotoxic, but low concentrations could activate the redox-sensitive antioxidant response element (ARE) via the Keap-1/Nrf2 redox switch, inducing genoprotective adaptations. Our objective was to test this hypothesis.

Methods

Peripheral lymphocytes from healthy volunteers were incubated for 30 minutes at 37°C in freshly prepared tea solutions (0.005, 0.01, 0.05%w/v (7, 14, 71 µmol/l total catechins) in phosphate buffered saline (PBS), with PBS as control) in the presence and absence of catalase (CAT). H₂O₂ in tea was measured colorimetrically. Oxidation-induced DNA lesions were measured by the Fpg-assisted comet assay.

Results

H₂O₂ concentrations in 0.005, 0.01, and 0.05% green tea after 30 minutes at 37°C were, respectively, ～3, ～7, and ～52 µmol/l. Cells incubated in 0.005 and 0.01% tea showed less (P < 0.001) DNA damage compared to control cells. Cells treated with 0.05% green tea showed ～50% (P < 0.001) more DNA damage. The presence of CAT prevented this damage, but did not remove the genoprotective effects of low-dose tea. No significant changes in expression of ARE-associated genes (HMOX1, NRF2, KEAP1, BACH1, and hOGG1) were seen in cells treated with tea or tea + CAT.

Conclusion

Genoprotection by low-dose green tea could be due to direct antioxidant protection by green tea polyphenols, or to H₂O₂-independent signalling pathways.     

Conservation of telomere protein complexes: shuffling through evolution

The rapid evolution of telomere proteins has hindered identification of orthologs from diverse species and created the impression that certain groups of eukaryotes have largely non-overlapping sets of telomere proteins. However, the recent identification of additional telomere proteins from various model organisms has dispelled this notion by expanding our understanding of the composition, architecture and range of telomere protein complexes present in individual species. It is now apparent that versions of the budding yeast CST complex and mammalian shelterin are present in multiple phyla. While the precise subunit composition and architecture of these complexes vary between species, the general function is often conserved. Despite the overall conservation of telomere protein complexes, there is still considerable species-specific variation, with some organisms having lost a particular subunit or even an entire complex. In some cases, complex components appear to have migrated between the telomere and the telomerase RNP. Finally, gene duplication has created telomere protein paralogs with novel functions. While one paralog may be part of a conserved telomere protein complex and have the expected function, the other paralog may serve in a completely different aspect of telomere biology.     

Molecular Characterization and Alternative Splicing of a Sodium Channel and DSC1 Ortholog Genes in Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)

Alternative splicing greatly contributes to the structural and functional diversity of voltage-gated sodium channels (VGSCs) by generating various isoforms with unique functional and pharmacological properties. Here, we identified a new optional exon 23 located in the linker between domains II and III, and four mutually exclusive exons (exons 27A, 27B, 27C, and 27D) in domains IIIS3 and IIIS4 of the sodium channel of Liposcelis bostrychophila (termed as LbVGSC). This suggested that more alternative splicing phenomena remained to be discovered in VGSCs. Inclusion of exon 27C might lead to generation of non-functional isoforms. Meanwhile, identification of three alternative exons (exons 11, 13A, and 13B), which were located in the linker between domains II and III, indicated that abundant splicing events occurred in the DSC1 ortholog channel of L. bostrychophila (termed as LbSC1). Exons 13A and 13B were generated by intron retention, and the presence of exon 13B relied on the inclusion of exon 13A. Exon 13B was specifically expressed in the embryonic stage and contained an in-frame stop codon, inclusion of which led to generation of truncated proteins with only the first two domains. Additionally, several co-occurring RNA editing events were identified in LbSC1. Furthermore, remarkable similarity between the structure and expression patterns of LbVGSC and LbSC1 were discovered, and a closer evolutionary relationship between VGSCs and DSC1 orthologs was verified. Taken together, the data provided abundant molecular information on VGSC and DSC1 orthologs in L. bostrychophila, a representative Psocoptera storage pest, and insights into the alternative splicing of these two channels.     

Indices of oxidative stress in pregnancy with fetal growth restriction

Intrauterine fetal growth restriction (IUGR), the main cause of premature delivery and fetal mortality, has been suggested to involve oxidative stress. We found elevated values of indices of oxidative stress in the blood serum of pregnant women with IUGR: increased levels of malondialdehyde and 4-hydroxyalkenals, decreased activity of α-1-antitrypsin and decreased total antioxidant capacity of the serum, with respect to healthy pregnancy. Twenty day treatment with 3 g of l-arginine and 75 mg of acetylsalicylic acid daily resulted in a decrease of the level of lipid peroxidation products and augmentation of α-1-antitrypsin activity. This study confirms the occurrence of oxidative stress in IUGR and demonstrates the beneficial effect of arginine/acetylsalicylic acid therapy in reducing oxidative stress in IUGR.     

Regulation of DNA glycosylases and their role in limiting disease

Abstract

This review will present a current understanding of mechanisms for the initiation of base excision repair (BER) of oxidatively-induced DNA damage and the biological consequences of deficiencies in these enzymes in mouse model systems and human populations.