2-methoxyestradiol enhances p53 protein transduction therapy-associated inhibition of the proliferation of oral cancer cells through the suppression of NFkappaB activity.

Protein transduction therapy using poly-arginine peptide can deliver the biologically active proteins. A previous study showed that 11 poly-arginine fused p53 protein (11R-p53) effectively penetrated across the plasma membrane and inhibited the proliferation of oral cancer cells. However, the intracellular half-life of the delivered protein was less than 36 h. Previous studies also showed that 2-methoxyestradiol (2-ME), an endogenous non-toxic estrogenic metabolite, induces the stabilization of the wild-type p53 protein in human cancer cells posttranscriptionally. In the present study, we examined whether 2-ME induced the stabilization of 11R-p53 and had an inhibitory effect on the proliferation of oral cancer cells. The application of 2-ME significantly enhanced the inhibitory effect of 11R-p53 on the proliferation of oral cancer cells. However, 2-ME had no effect on the intracellular half-life of 11R-p53 in oral cancer cells. Of interest is the finding that 2-ME suppressed the transcriptional activity of NFkappaB, which has an important role in tumorigenesis, but did not affect p53 transcriptional activity. These results suggest that 2-ME synergistically enhances the 11R-p53-induced inhibition of the proliferation of oral cancer cells through the suppression of NFkB transcription.</p

Exposure of mouse to high gravitation forces induces long-term potentiation in the hippocampus.

The central nervous system is highly plastic and has been shown to undergo both transient and chronic adaptive changes in response to environmental influences. The purpose of this study was to investigate the effect of hypergravic field on long-term potentiation (LTP) in the mouse hippocampus. Exposure of mice to 4G fields for 48 h had no effect on input-output coupling during extracellular stimulation of Schaffer collaterals and paired pulse facilitation, suggesting that the hypergravic exposure had no detrimental effect on basal neurotransmission in the hippocampus. However, the exposure to 4G fields for 48 h significantly induced LTP compared with the control mouse hippocampus. In contrast, no significant changes of late-phase LTP (L-LTP) were found in the hippocampi of mice exposed to the hypergravic field. Exposure of mice to 4G fields for 48 h enhanced AMPA receptor phosphorylation but not cyclic AMP-responsive element binding protein (CREB) phosphorylation. These results suggest that exposure to hyperdynamic fields influences the synaptic plasticity in the hippocampus.</p

Regulation of Mitochondrial Dynamics and Neurodegenerative Diseases

Mitochondria are important cellular organelles in most metabolic processes and have a highly dynamic nature, undergoing frequent fission and fusion. The dynamic balance between fission and fusion plays critical roles in mitochondrial functions. In recent studies, several large GTPases have been identified as key molecular factors in mitochondrial fission and fusion. Moreover, the posttranslational modifications of these large GTPases, including phosphorylation, ubiquitination and SUMOylation, have been shown to be involved in the regulation of mitochondrial dynamics. Neurons are particularly sensitive and vulnerable to any abnormalities in mitochondrial dynamics, due to their large energy demand and long extended processes. Emerging evidences have thus indicated a strong linkage between mitochondria and neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and Huntington's disease. In this review, we will describe the regulation of mitochondrial dynamics and its role in neurodegenerative diseases

Colocalization of oxytocin and phosphorylated form of elongation factor 2 in the rat hypothalamus

Oxytocin (OT) is one of the neuropituitary hormones and is synthesized in the neurons of the paraventricular nucleus (PVN) and supraoptic nucleus (SON). Previous studies have shown that the mRNAs encoding OT are delivered from the soma to both dendrites and axons of the neurons in the PVN and SON. However, it has not been elucidated whether a translational regulation mechanism to enable local synthesis of the hormone exists in the axons of the neurons of PVN and SON. Elongation factor 2 (EF2) is essential for polypeptide synthesis during protein translation. Moreover, phosphorylation of EF2 by EF2 kinase enhances the translation of certain mRNA species. In the present study, in order to shed light on the mechanisms involved in the translational regulation of OT synthesis, we investigated the localization of phosphorylated EF2. Phospho-EF2 was localized in the soma of the neurons in PVN and SON, and in the swellings of the median eminence where axonal tracts of the neurons in the PVN and SON exist. The phosphorylated form was also observed in the rat hypophysis. Moreover, phospho-EF2 and OT were colocalized in a part of the neurons in the PVN and SON. These results suggest that OT may be partially translated in the axons of neurons in the PVN and SON, and then secreted from the pituitary

Deletion of Long Isoform of Eukaryotic Elongation Factor 1Bδ Leads to Audiogenic Seizures and Aversive Stimulus-Induced Long-Lasting Activity Suppression in Mice

Alternative splicing enables a gene to give rise to diverse protein products. The Eef1d gene produces two isoforms: a short isoform that encodes translation elongation factor 1Bδ (eEF1Bδ1), and a long isoform that encodes the heat shock-responsive transcription factor eEF1BδL. Previously, we found that eEF1BδL was a splice variant that was specific to the brain and testis, and the protein encoded is thought to have a function in the central nervous system. In this study, we generated knockout (KO) mice of C57BL/6J background that selectively lacked a specific exon in Eef1d for the long isoform. These KO mice lacked eEF1BδL, but not eEF1Bδ1, in the brain. Although the KO mice showed normal anxiety-related and learning behavior in behavioral tests, some showed severe seizures in response to loud sounds (90 dBA), an audiogenic seizures (AGS) response. Furthermore, after the KO mice had been subjected to the fear conditioning test, they showed remarkably decreased locomotor activity in their home cage and in the open-field and elevated plus-maze tests. After the fear conditioning test, a significant decrease in brain weight, atrophy of the hippocampus and midbrain, and reduced cortical layer thickness were observed in the KO mice. We also found a compensatory increase in the eEF1Bδ1 level and elevated protein synthesis with the induction of endoplasmic reticulum stress markers in these mice. Our results suggest that eEF1BδL has an important role in normal brain function especially when exposed to external stimuli

CaM kinase Iα–induced phosphorylation of Drp1 regulates mitochondrial morphology

Mitochondria are dynamic organelles that frequently move, divide, and fuse with one another to maintain their architecture and functions. However, the signaling mechanisms involved in these processes are still not well characterized. In this study, we analyze mitochondrial dynamics and morphology in neurons. Using time-lapse imaging, we find that Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) causes a rapid halt in mitochondrial movement and induces mitochondrial fission. VDCC-associated Ca2+ signaling stimulates phosphorylation of dynamin-related protein 1 (Drp1) at serine 600 via activation of Ca2+/calmodulin-dependent protein kinase Iα (CaMKIα). In neurons and HeLa cells, phosphorylation of Drp1 at serine 600 is associated with an increase in Drp1 translocation to mitochondria, whereas in vitro, phosphorylation of Drp1 results in an increase in its affinity for Fis1. CaMKIα is a widely expressed protein kinase, suggesting that Ca2+ is likely to be functionally important in the control of mitochondrial dynamics through regulation of Drp1 phosphorylation in neurons and other cell types

Combining poly-arginine with the hydrophobic counter-anion 4-(1-pyrenyl)-butyric acid for protein transduction in transdermal delivery

Topical therapy is the most favored form of treatment for whitening against hyperpigmentation and sunburn because it lends itself to self-administration, patient compliance, and absence of systemic adverse effects. However, transdermal delivery of hydrophilic chemicals is difficult. The main purpose of this study is to develop a delivering system of hydrophilic drugs and proteins across the skin. Hydroquinone (HQ), a well-known tyrosinase inhibitor and antimelanogenesis compound, and enhanced green fluorescent protein (EGFP) were fused with eleven poly-arginine (11R). Both HQ-11R and EGFP-11R were efficiently delivered in B16 cells, a mouse melanoma cell line. HQ-11R was as effective as HQ alone at inhibiting melanin synthesis in B16 cells. EGFP-11R was efficiently delivered into cells of the epidermis with 4-(1-pyrenyl)-butyric acid (PB), a counteranion bearing an aromatic hydrophobic moiety, in vivo, but EGFP alone or EGFP-11R without PB was not. Finally, topical application of HQ-11R with PB significantly inhibited UV irradiation-induced pigmentation in guinea pigs compared with HQ alone. These results suggest that topical therapy using poly-arginine in combination with PB is useful for the delivery of hydrophilic drugs and proteins by the transdermal route