Mitochondrial Dysfunction in Pten Haplo-Insufficient Mice with Social Deficits and Repetitive Behavior: Interplay between Pten and p53

Etiology of aberrant social behavior consistently points to a strong polygenetic component involved in fundamental developmental pathways, with the potential of being enhanced by defects in bioenergetics. To this end, the occurrence of social deficits and mitochondrial outcomes were evaluated in conditional Pten (Phosphatase and tensin homolog) haplo-insufficient mice, in which only one allele was selectively knocked-out in neural tissues. Pten mutations have been linked to Alzheimer's disease and syndromic autism spectrum disorders, among others. By 4-6 weeks of age, Pten insufficiency resulted in the increase of several mitochondrial Complex activities (II-III, IV and V) not accompanied by increases in mitochondrial mass, consistent with an activation of the PI3K/Akt pathway, of which Pten is a negative modulator. At 8-13 weeks of age, Pten haplo-insufficient mice did not show significant behavioral abnormalities or changes in mitochondrial outcomes, but by 20-29 weeks, they displayed aberrant social behavior (social avoidance, failure to recognize familiar mouse, and repetitive self-grooming), macrocephaly, increased oxidative stress, decreased cytochrome c oxidase (CCO) activity (50%) and increased mtDNA deletions in cerebellum and hippocampus. Mitochondrial dysfunction was the result of a downregulation of p53-signaling pathway evaluated by lower protein expression of p21 (65% of controls) and the CCO chaperone SCO2 (47% of controls), two p53-downstream targets. This mechanism was confirmed in Pten-deficient striatal neurons and, HCT 116 cells with different p53 gene dosage. These results suggest a unique pathogenic mechanism of the Pten-p53 axis in mice with aberrant social behavior: loss of Pten (via p53) impairs mitochondrial function elicited by an early defective assembly of CCO and later enhanced by the accumulation of mtDNA deletions. Consistent with our results, (i) SCO2 deficiency and/or CCO activity defects have been reported in patients with learning disabilities including autism and (ii) mutated proteins in ASD have been found associated with p53-signaling pathways.     

Intracellular delivery of glutathione S-transferase-fused proteins into mammalian cells by polyethylenimine-glutathione conjugates

The glutathione S-transferase (GST)-fused protein expression system has been extensively used to generate a large quantity of proteins and has served for functional analysis in vitro. In this study, we developed a novel approach for the efficient intracellular delivery of GST-fused proteins into living cells to expand their usefulness up to in vivo use. Since protein cationization techniques are powerful strategies for efficient intracellular uptake by adsorptive-mediated endocytosis, GST-fused proteins were cationized by forming a complex with a polycationic polyethylenimine (PEI)-glutathione conjugate. On screening of protein transduction, optimized PEI-glutathione conjugate for protein transduction was characterized by a partly oligomerized mixture of PEI with average molecular masses of 600 (PEI600) modified with multiple glutathiones, which could have sufficient avidity for GST. Furthermore, enhanced endosomal escape of transduced GST-fused proteins was observed when they were delivered with a glutathione-conjugated PEI600 derivative possessing a hydroxybutenyl moiety. These results were confirmed by both intracellular confocal imaging of GST-fused green fluorescent protein and activation of an endogenous growth signal transduction pathway by a GST-fused constitutively active mutant of a kinase protein. These PEI-glutathione conjugates seem to be convenient molecular tools for protein transduction of widely used GST-fused proteins.     

A case report on human type B botulism

A case of type B human botulism was found in Tochigi Prefecture in November, 1984. Botulinum type B toxin was detected in the serum and feces of the patient. The serum toxin was activated by trypsinization. Type B toxin was demonstrated in cooked meat medium cultures of the fecal specimens. The patient recovered after administration of type A, B, E and F quadrivalent antitoxin.     

Oxygen concentration-dependent induction of a 140-kDa protein in magnetic bacterium Magnetospirillum magnetotacticum MS-1

Abstract The magnetic bacterium Magnetospirillum magnetotacticum prefers a microaerobic habitat and should be able to sense oxygen. Therefore, the bacterium was cultured under atmospheres containing 0–5% O₂ and analyzed for oxygen-dependent changes in the levels of its protein components by sodium dodecyl sulfate-polyccrylamide gel electrophoresis (SDS-PAGE). The analysis revealed a marked anaerobic induction of a 140-kDa protein, which was suppressed when M. magnetotacticum was switched from microaerobic (<1% O₂) to aerobic (>1% O₂) growth conditions. Although its function remains to be determined, the 140-kDa protein may serve as a useful tool to gain insight into the physiology of the organism.     

Enhanced skeletal muscle glycogen repletion after endurance exercise is associated with higher plasma insulin and skeletal muscle hexokinase 2 protein levels in mice: comparison of level running and downhill running model

Journal of Physiology and Biochemistry - To identify factors that influence post-exercise muscle glycogen repletion, we compared the glycogen recovery after level running with downhill running, an...     

Long-Duration Carbon Dioxide Anesthesia of Fish Using Ultra Fine (Nano-Scale) Bubbles

Introduction: We investigated whether adding ultrafine (nano-scale) oxygen-carrying bubbles to water concurrently with dissolved carbon-dioxide (CO₂) could result in safe, long-duration anesthesia for fish. Results: To confirm the lethal effects of CO₂ alone, fishes were anesthetized with dissolved CO₂ in 20°C seawater. Within 30 minutes, all fishes, regardless of species, died suddenly due to CO₂-induced narcosis, even when the water was saturated with oxygen. Death was attributed to respiration failure caused by hypoxemia. When ultrafine oxygen-carrying bubbles were supplied along with dissolved CO₂, five chicken grunts were able to remain anesthetized for 22 hours and awoke normally within 2–3 hours after cessation of anesthesia. Conclusions: The high internal pressures and oxygen levels of the ultrafine bubbles enabled efficient oxygen diffusion across the branchia and permitted the organismal oxygen demands of individual anesthetized fish to be met. Thus, we demonstrated a method for safe, long-duration carbon dioxide anesthesia in living fish under normal water temperatures.     

Specific arrangements of cortical microtubules are correlated with the architecture ofmeristems in shoot apices of angiosperms and gymnosperms

Arrangements of corticalmicrotubules (MTs) as seen in median longitudinal cryosections of shoot apices of several angiosperms and gymnosperms were studied by indirect immunofluorescence microscopy.Bryophyllum, Clethra, Helianthus, Houttuynia, Vinca (angiosperms), andPinus, Cedrus, Cedrus andGinkgo (gymnosperms) were examined. In all angiosperm apices collected during the growing season, MTs were mainly arranged anticlinally in the tunica, randomly in the corpus, and transversely in the rib meristem. This pattern of arrangements of MTs was further confirmed by electron microscopy inBryophyllum apices. In the apices of winter shoots MTs in the rib meristem were arranged randomly, indicating a seasonal change with respect to their arrangment. In all examined gymnosperm apices, populations of superficial cells showed both random and anticlinal arrangements of MTs, in contrast to those of angiosperm apices that consistently show anticlinally arranged MTs. In the shoot apices of both angiosperms and gymnosperms, cortical MTs were arranged perpendicularly to the directions of cell expansion. The significance of MTs in the maintrnance of the different architectures of shoot apices in angiosperms and gymnosperms is discussed.     

Three-dimensional structural model analysis of the binding site of lithocholic acid, an inhibitor of DNA polymerase beta and DNA topoisomerase II.

The molecular action of lithocholic acid (LCA), a selective inhibitor of mammalian DNA polymerase beta (pol beta), was investigated. We found that LCA could also strongly inhibit the activity of human DNA topoisomerase II (topo II). No other DNA metabolic enzymes tested were affected by LCA. Therefore, LCA should be classified as an inhibitor of both pol beta and topo II. Here, we report the molecular interaction of LCA with pol beta and topo II. By three-dimensional structural model analysis and by comparison with the spatial positioning of specific amino acids binding to LCA on pol beta (Lys60, Leu77, and Thr79), we obtained supplementary information that allowed us to build a structural model of topo II. Modeling analysis revealed that the LCA-interaction interface in both enzymes has a pocket comprised of three amino acids in common, which binds to the LCA molecule. In topo II, the three amino acid residues were Lys720, Leu760, and Thr791. These results suggested that the LCA binding domains of pol beta and topo II are three-dimensionally very similar.