PI3 kinase/Akt activation mediates estrogen and IGF-1 nigral DA neuronal neuroprotection against a unilateral rat model of Parkinson's disease

Recently, using the medial forebrain bundle (MFB) 6-hydroxydopmaine (6-OHDA) lesion rat model of Parkinson's disease (PD), we have demonstrated that blockade of central IGF-1 receptors (IGF-1R) attenuated estrogen neuroprotection of substantia nigra pars compacta (SNpc) DA neurons, but exacerbated 6-OHDA lesions in IGF-1 only treated rats (Quesada and Micevych [2004]: J Neurosci Res 75:107-116). This suggested that the IGF-1 system is a central mechanism through which estrogen acts to protect the nigrostriatal DA system. Moreover, these results also suggest that IGF-1R-induced intracellular signaling pathways are involved in the estrogen mechanism that promotes neuronal survival. In vitro, two convergent intracellular signaling pathways used by estrogen and IGF-1, the mitogen-activated protein kinase (MAPK/ERK), and phosphatidyl-inositol-3-kinase/Akt (PI3K/Akt), have been demonstrated to be neuroprotective. Continuous central infusions of MAPK/ERK and PI3K/Akt inhibitors were used to test the hypothesis that one or both of these signal transduction pathways mediates estrogen and/or IGF-1 neuroprotection of SNpc DA neurons after a unilateral administration of 6-OHDA into the MFB of rats. Motor behavior tests and tyrosine hydroxylase immunoreactivity revealed that the inhibitor of the PI3K/Akt pathway (LY294002) blocked the survival effects of both estrogen and IGF-1, while an inhibitor of the MAPK/ERK signaling (PD98059) was ineffective. Western blot analyses showed that estrogen and IGF-1 treatments increased PI3K/Akt activation in the SN; however, MAPK/ERK activation was decreased in the SN. Indeed, continuous infusions of inhibitors blocked phosphorylation of PI3K/Akt and MAPK/ERK. These findings indicate that estrogen and IGF-1-mediated SNpc DA neuronal protection is dependent on PI3K/Akt signaling, but not on the MAPK/ERK pathway.     

Ribonuclease-neutralized pancreatic microsomal membranes from livestock for in vitro co-translational protein translocation

Here, we demonstrate that pancreatic microsomal membranes from pigs, sheep, or cattle destined for human consumption can be used as a valuable and ethically correct alternative to dog microsomes for cell-free protein translocation. By adding adequate ribonuclease (RNase) inhibitors to the membrane fraction, successful in vitro co-translational translocation of wild-type and chimeric pre-prolactin into the lumen of rough microsomes was obtained. In addition, the human type I integral membrane proteins CD4 and VCAM-1 were efficiently glycosylated in RNase-treated microsomes. Thus, RNase-neutralized pancreatic membrane fractions from pig, cow, or sheep are a cheap, easily accessible, and fulfilling alternative to canine microsomes.     

Evaluation of hypoglycemic therapeutics and nutritional supplementation for type 2 diabetes mellitus management: An insight on molecular approaches

Biotechnology Letters - This review aims to summarize the current management of type 2 diabetes principles, including oral hypoglycemic agents, types of insulin administration, diet maintenance,...     

Nox1-dependent reactive oxygen generation is regulated by Rac1

Rac1 has been implicated in the generation of reactive oxygen species (ROS) in several cell types, but the enzymatic origin of the ROS has not been proven. The present studies demonstrate that Nox1, a homolog of the phagocyte NADPH-oxidase component gp91(phox), is activated by Rac1. When Nox1 is co-expressed along with its regulatory subunits NOXO1 and NOXA1, significant ROS generation is seen. Herein, co-expression of constitutively active Rac1(G12V), but not wild-type Rac1, resulted in marked further stimulation of activity. Decreased Rac1 expression using small interfering RNA reduced Nox1-dependent ROS. CDC42(G12V) failed to increase activity, and small interfering RNA directed against CDC42 failed to decrease activity, pointing to specificity for Rac. TPR domain mutants of NOXA1 that interfere with Rac1 binding were ineffective in supporting Nox1-dependent ROS generation. Immunoprecipitation experiments demonstrated a complex containing Rac1(G12V), NOXO1, NOXA1, and Nox1. CDC42(G12V) could not substitute for Rac1(G12V) in such a complex. Nox1 formed a complex with Rac1(G12V) that was independent of NOXA1 and NOXO1, consistent with direct binding of Rac1(G12V) to Nox1. Rac1(G12V) interaction with NOXA1 was enhanced by Nox1 and NOXO1, suggesting cooperative binding. A model is presented comparing activation by regulatory subunits of Nox1 versus gp91(phox) (Nox2) in which Rac1 activation provides a major trigger that acutely activates Nox1-dependent ROS generation.     

Heritable endosymbionts of Drosophila

Although heritable microorganisms are increasingly recognized as widespread in insects, no systematic screens for such symbionts have been conducted in Drosophila species (the primary insect genetic models for studies of evolution, development, and innate immunity). Previous efforts screened relatively few Drosophila lineages, mainly for Wolbachia. We conducted an extensive survey of potentially heritable endosymbionts from any bacterial lineage via PCR screens of mature ovaries in 181 recently collected fly strains representing 35 species from 11 species groups. Due to our fly sampling methods, however, we are likely to have missed fly strains infected with sex ratio-distorting endosymbionts. Only Wolbachia and Spiroplasma, both widespread in insects, were confirmed as symbionts. These findings indicate that in contrast to some other insect groups, other heritable symbionts are uncommon in Drosophila species, possibly reflecting a robust innate immune response that eliminates many bacteria. A more extensive survey targeted these two symbiont types through diagnostic PCR in 1225 strains representing 225 species from 32 species groups. Of these, 19 species were infected by Wolbachia while only 3 species had Spiroplasma. Several new strains of Wolbachia and Spiroplasma were discovered, including ones divergent from any reported to date. The phylogenetic distribution of Wolbachia and Spiroplasma in Drosophila is discussed.     

NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region

The small intestinal BB Na⁺/H⁺ antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P₃) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His₆ proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475–589), F2 (amino acids 590–667), F3 (amino acids 668–747), and F4 (amino acids 748–832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P₂ and PI(3,4,5)P₃ bound only to the NHE3 F1 fusion protein (amino acids 475–589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na⁺/H⁺ exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P₂ and PI(3,4,5)P₃ binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P₂ and PI(3,4,5)P₃ binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P₂ or PI(3,4,5)P₃ binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr⁵⁰¹–Arg⁵¹² and Arg⁵²⁰–Arg⁵⁵²) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.