Inhibition of mitochondrial function in astrocytes: implications for neuroprotection

Much evidence suggests that astrocytes protect neurons against ischemic injury. Although astrocytes are more resistant to some insults than neurons, few studies offer insight into the real time changes of astrocytic protective functions with stress. Mitochondria are one of the primary targets of ischemic injury in astrocytes. We investigated the time course of changes in astrocytic ATP levels, plasma membrane potential, and glutamate uptake, a key protective function, induced by mitochondrial inhibition. Our results show that significant functional change precedes reduction in astrocytic viability with mitochondrial inhibition. Using the mitochondrial inhibitor fluorocitrate (FC, 0.25 mmol/L) that is preferentially taken by astrocytes we found that inhibition of astrocyte mitochondria increased vulnerability of co-cultured neurons to glutamate toxicity. In our studies, the rates of FC-induced astrocytic mitochondrial depolarization were accelerated in mixed astrocyte/neuron cultures. We hypothesized that the more rapid mitochondrial depolarization was promoted by an additional energetic demand imposed be the co-cultured neurons. To test this hypothesis, we exposed pure astrocytic cultures to 0.01-1 mmol/L aspartate as a metabolic load. Aspartate application accelerated the rates of FC-induced mitochondrial depolarization, and, at 1 mmol/L, induced astrocytic death, suggesting that strong energetic demands during ischemia can compromise astrocytic function and viability.     

Comparison of Ionized Calcium-binding Adapter Molecule 1 Immunoreactivity of the Hippocampal Dentate Gyrus and CA1 Region in Adult and Aged Dogs

Similarities between age-related changes in the canine and human brain have resulted in the general acceptance of the canine brain as a model of human brain aging. The hippocampus is essentially required for intact cognitive ability and appears to be particularly vulnerable to the aging process. We observed changes in ionized calcium-binding adapter molecule 1 (Iba-1, a microglial marker) immunoreactivity and protein levels in the hippocampal dentate gyrus and CA1 region of adult (2-3 years) and aged (10-12 years) dogs. We also observed the interferon-gamma (IFN-gamma), a pro-inflammatory cytokine, protein levels in these groups. In the dentate gyrus and CA1 region of the adult dog, Iba-1 immunoreactive microglia were well distributed and their processes were highly ramified. However, in the aged dog, the processes of Iba-1 immunoreactive microglia were hypertrophied in the dentate gyrus. Moreover, Iba-1 protein level in the dentate gyrus in the aged dog was higher than in the adult dog. IFN-gamma expression was increased in the dentate gyrus homogenates of aged dogs than adult dogs. In addition, we found that some neurons were positive to Fluoro-Jade B (a marker for neuronal degeneration) in the dentate polymorphic layer, but not in the hippocampal CA1 region in the aged dog. These results suggest that Iba-1 immunoreactive microglia are hypertrophied in the dentate gyrus in the aged dog.     

Immunological dynamics in response to two anthrax vaccines in mice

In order to understand the variation of humoral and cellular immune responses to A16R live spore and AVA vaccine and to identify efficient immunological parameters for the early evaluation of post immu- nization in mice, we dynamically monitored the antibody production and cellular responses after the vaccination of Balb/C mice with the anthrax vaccines. The results show that both anti-AVA and anti-Spore antibodies were detectable in the A16R live spore vaccinated group while high titers of anti-AVA antibodies but not anti-Spore antibodies existed in the AVA-immunized group. IgG1 and IgG2 were the major subtypes of IgG in both of the two groups. However, the IgG2a level was significantly higher in the A16R group than in the AVA group. At the cellular level, responses of antigen-specific TH2, TH1 and plasma cells were detected. The peripheral TH2 responses could be seen on day 5 after vac- cination, and remained at a high level throughout the experiment (from day 5 post primary immuniza- tion to day 60 post the tertiary immunization); the TH1 responses to A16R vaccine appeared on day 5, while the responses to AVA could only be detected by day 7 after the secondary immunization; a low level of TH1 responses could be observed at the end of the experiment. Antigen-specific plasma cells could be found in the peripheral blood of both the immunized groups, however, the responses in the A16R group appeared earlier, lasted longer, and shown an ascending tendency until the end of the ex- periment when the plasma cell responses in the AVA group were reduced to a very low level. The re- sults suggest that the multiple antigen containing A16R live spore vaccine induces better immune re- sponses than AVA. Combined with serum antibody titers, TH2, TH1 and plasma cell responses could be used as immunological parameters for the evaluation of vaccine efficacy. These findings may afford new insight into the early evaluation of vaccination as well as being a powerful strategy for vaccine development.     

v-Crk induces Rac-dependent membrane ruffling and cell migration in CAS-deficient embryonic fibroblasts

Crk-associated substrate (CAS) is a focal adhesion protein that is involved in integrin signaling and cell migration. CAS deficiency reduces the migration and spreading of cells, both of which are processes mediated by Rac activation. We examined the functions of v-Crk, the oncogene product of the CT10 virus p47gag-crk, which affects cell migration and spreading, membrane ruffling, and Rac activation in CAS-deficient mouse embryonic fibroblasts (CAS-/- MEFs). CAS-/- MEFs showed less spreading than did CAS+/+ MEFs, but spreading was recovered in mutant cells that expressed v-Crk (CAS-/-v-Crk MEF). We observed that the reduction in spreading was linked to the formation of membrane ruffles, which were accompanied by Rac activation. In CAS-/- MEFs, Rac activity was significantly reduced, and Rac was not localized to the membrane. In contrast, Rac was active and localized to the membrane in CAS-/-v-Crk MEFs. Lamellipodia protrusion and ruffle retraction velocities were both reduced in CAS-/- MEFs, but not in CAS-/-v-Crk MEFs. We also found that microinjection of anti-gag antibodies inhibited the migration of CAS-/-v-Crk MEFs. These findings indicate that v-Crk controls cell migration and membrane dynamics by activating Rac in CAS-deficient MEFs.     

Complete nucleotide sequence and organization of the mitogenome of the silk moth Caligula boisduvalii (Lepidoptera: Saturniidae) and comparison with other lepidopteran insects

The 15,360-bp long complete mitogenome of Caligula boisduvalii possesses a gene arrangement and content identical to other completely sequenced lepidopteran mitogenomes, but different from the common arrangement found in most insect order, as the result of the movement of tRNA(Met) to a position 5'-upstream of tRNA Ile. The 330-bp A+T-rich region is apparently capable of forming a stem-and-loop structure, which harbors the conserved flanking sequences at both ends. Dissimilar to what has been seen in other sequenced lepidopteran insects, the initiation codon for C. boisduvalii COI appears to be TTG, which is a rare, but apparently possible initiation codon. The ATP8, ATP6, ND4L, and ND6 genes, which neighbor another PCG at their 3' end, all harbored potential sequences for the formation of a hairpin structure. This is suggestive of the importance of such structures for the precise cleavage of the mRNA of mature PCGs. Phylogenetic analyses of available sequenced species of Bombycoidea, Pyraloidea, and Tortricidea supported the morphology-based current hypothesis that Bombycoidea and Pyraloidea are monophyletic (Obtectomera). As previously suggested, Bombycidae (Bombyx mori and B. mandarina) and Saturniidae (Antheraea pernyi and C. boisduvalii) formed a reciprocal monophyletic group.     

MicroRNA miR-199a* regulates the MET proto-oncogene and the downstream extracellular signal-regulated kinase 2 (ERK2)

MicroRNAs (miRNAs) constitute a class of small noncoding RNAs that play important roles in a variety of biological processes including development, apoptosis, proliferation, and differentiation. Here we show that the expression of miR-199a and miR-199a* (miR-199a/a*), which are processed from the same precursor, is confined to fibroblast cells among cultured cell lines. The fibroblast-specific expression pattern correlated well with methylation patterns: gene loci on chromosome 1 and 19 were fully methylated in all examined cell lines but unmethylated in fibroblasts. Transfection of miR-199a and/or -199a* mimetics into several cancer cell lines caused prominent apoptosis with miR-199a* being more pro-apoptotic. The mechanism underlying apoptosis induced by miR-199a was caspase-dependent, whereas a caspase-independent pathway was involved in apoptosis induced by miR-199a* in A549 cells. By employing microarray and immunoblotting analyses, we identified the MET proto-oncogene as a target of miR-199a*. Studies with a luciferase reporter fused to the 3'-untranslated region of the MET gene demonstrated miR-199a*-mediated down-regulation of luciferase activity through a binding site of miR-199a*. Interestingly, extracellular signal-regulated kinase 2 (ERK2) was also down-regulated by miR-199a*. Coordinated down-regulation of both MET and its downstream effector ERK2 by miR-199a* may be effective in inhibiting not only cell proliferation but also motility and invasive capabilities of tumor cells.     

Silencing of NtMPK4 impairs CO-induced stomatal closure, activation of anion channels and cytosolic Casignals in Nicotiana tabacum guard cells

Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of Nt MPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of Nt MPK4-silenced plants do not close in elevated atmospheric CO₂, and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO₂ activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in Nt MPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca²⁺ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca²⁺ than in those with steady Ca²⁺ levels. Both the darkness-induced anion channel activation and Ca²⁺ signals were repressed in Nt MPK4-silenced guard cells. These data show that CO₂ and darkness can activate anion channels in a Ca²⁺-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca²⁺ concentration. Nt MPK4 plays an essential role in CO₂- and darkness-induced activation of guard-cell anion channels, through Ca²⁺-independent as well as Ca²⁺-dependent signalling pathways.     

Selenoproteins mediate T cell immunity through an antioxidant mechanism

Selenium is an essential dietary element with antioxidant roles in immune regulation, but there is little understanding of how this element acts at the molecular level in host defense and inflammatory disease. Selenium is incorporated into the amino acid selenocysteine (Sec), which in turn is inserted into selenoproteins in a manner dependent on Sec tRNA([Ser]Sec). To investigate the molecular mechanism that links selenium to T cell immunity, we generated mice with selenoprotein-less T cells by cell type-specific ablation of the Sec tRNA([Ser]Sec) gene (trsp). Herein, we show that these mutant mice exhibit decreased pools of mature T cells and a defect in T cell-dependent antibody responses. We also demonstrate that selenoprotein deficiency leads to oxidant hyperproduction in T cells and thereby suppresses T cell proliferation in response to T cell receptor stimulation. These findings offer novel insights into immune function of selenium and physiological antioxidants.     

Ginsenosides Rb1 and Rg1 suppress triglyceride accumulation in 3T3-L1 adipocytes and enhance beta-cell insulin secretion and viability in Min6 cells via PKA-dependent pathways

Ginseng root is known to induce anti-diabetic activity, but the key components involved are unknown. We investigated which major ginsenosides in ginseng enhanced glucose homeostasis by in vitro studies. Rb1 and Rg1 reduced the triglyceride accumulation in 3T3-L1 adipocytes by activating PKA with increased intracellular cAMP. However, the insulin-stimulated glucose uptake was enhanced by Rb1 and Rg1 via activation of phosphatidylinositol-3 kinase. Rb1 and Rg1 promoted glucose-stimulated insulin secretion and cell viability in Min6 cells through PKA which augmented IRS2 expression to enhance insulin/IGF-1 signaling. These results suggest that Rb1 and Rg1 improved glucose homeostasis through the activation of a PKA like glucagon-like peptide-1 receptor agonist.     

A micellar prodrug of paclitaxel conjugated to cyclotriphosphazene

A novel water soluble and biodegradable cyclotriphosphazene-paclitaxel conjugate was prepared by reacting 2'-succinyl paclitaxel with cyclotriphosphazenes bearing equimolar glycyl-L-lysine and methoxy poly(ethylene glycol) as side groups. The macromolecular conjugate was found to self-assemble in aqueous solution to form stable micelles with a mean hydrodynamic diameter of 24.7 nm and a low critical micelle concentration of 10 mg/L. The present conjugate exhibited lower than free paclitaxel but reasonably high in vitro cytotoxicity against selected human tumor cells due to their hydrolytic degradation in PBS solution.