From modulation of cellular plasticity to potentiation of therapeutic resistance: new and emerging roles of MYB transcription factors in human malignancies.

MYB transcription factors are encoded by a large family of highly conserved genes from plants to vertebrates. There are three members of the MYB gene family in human, namely, MYB, MYBL1, and MYBL2 that encode MYB/c-MYB, MYBL1/A-MYB, and MYBL2/B-MYB, respectively. MYB was the first member to be identified as a cellular homolog of the v-myb oncogene carried by the avian myeloblastosis virus (AMV) causing leukemia in chickens. Under the normal scenario, MYB is predominantly expressed in hematopoietic tissues, colonic crypts, and neural stem cells and plays a role in maintaining the undifferentiated state of the cells. Over the years, aberrant expression of MYB genes has been reported in several malignancies and recent years have witnessed tremendous progress in understanding of their roles in processes associated with cancer development. Here, we review various MYB alterations reported in cancer along with the roles of MYB family proteins in tumor cell plasticity, therapy resistance, and other hallmarks of cancer. We also discuss studies that provide mechanistic insights into the oncogenic functions of MYB transcription factors to identify potential therapeutic vulnerabilities.

MYB exhibits racially disparate expression, clinicopathologic association, and predictive potential for biochemical recurrence in prostate cancer.

MYB acts as a potentiator of aggressiveness and castration resistance in prostate cancer (PCa) through aberrant activation of androgen receptor (AR) signaling. Since Black men experience higher PCa incidence and mortality than White men, we examined if MYB was differentially expressed in prostate tumors from patients of these racial backgrounds. The data reveal that aberrant MYB expression starts early in precancerous high-grade prostate intraepithelial neoplastic lesions and increases progressively in malignant cells. PCa tissues from Black patients exhibit higher MYB expression than White patients in overall and grade-wise comparisons. MYB also exhibits a positive correlation with AR expression and both display higher expression in advanced tumor stages. Notably, we find that MYB is a better predictor of biochemical recurrence than AR, pre-treatment PSA, or Gleason's grades. These findings establish MYB as a promising molecular target in PCa that could be used for improved risk prediction and therapeutic planning.

Comparative study of the molecular mechanisms underlying the G protein and ß-arrestin-dependent pathways that lead to ERKs activation upon stimulation by dopamine D2 receptor.

Dopamine D2 receptor (D2 R) has been shown to activate extracellular signal-regulated kinases (ERKs) via distinct pathways dependent on either G-protein or ß-arrestin. However, there has not been a systematic study of the regulatory process of D2 R-mediated ERKs activation by G protein- versus ß-arrestin-dependent signaling since D2 R stimulation of ERKs reflects the simultaneous action of both pathways. Here, we investigated that differential regulation of D2 R-mediated ERKs activation via these two pathways. Our results showed that G protein-dependent ERKs activation was transient, rapid, reached maximum level at around 2 min, and importantly, the activated ERKs were entirely confined to the cytoplasm. In contrast, ß-arrestin-dependent ERKs activation was more sustained, slower, reached maximum level at around 10 min, and phosphorylated ERKs translocated into the nucleus. Src was found to be commonly involved in both the G protein- and ß-arrestin-dependent pathway-mediated ERKs activation. Pertussis toxin Gi/o inhibitor, GRK2-CT, AG1478 epidermal growth factor receptor inhibitor, and wortmannin phosphoinositide 3-kinase inhibitor all blocked G protein-dependent ERKs activation. In contrast, GRK2 and ß-Arr2 played a main role in ß-arrestin-dependent ERKs activation. Receptor endocytosis showed minimal effect on the activation of ERKs mediated by both pathways. Furthermore, we found that the formation of a complex composed of phospho-ERKs, ß-Arr2, and importinß1 promoted the nuclear translocation of activated ERKs. The differential regulation of various cellular components, as well as temporal and spatial patterns of ERKs activation via these two pathways, suggest the existence of distinct physiological outcomes.

Ubiquitination of GRK2 Is Required for the ß-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases.

A class-A GPCR dopamine D2 receptor (D2R) plays a critical role in the proper functioning of neuronal circuits through the downstream activation of both G-protein- and ß-arrestin-dependent signaling pathways. Understanding the signaling pathways downstream of D2R is critical for developing effective therapies with which to treat dopamine (DA)-related disorders such as Parkinson's disease and schizophrenia. Extensive studies have focused on the regulation of D2R-mediated extracellular-signal-regulated kinase (ERK) 1/2 signaling; however, the manner in which ERKs are activated upon the stimulation of a specific signaling pathway of D2R remains unclear. The present study conducted a variety of experimental techniques, including loss-of-function experiments, site-directed mutagenesis, and the determination of protein interactions, in order to investigate the mechanisms underlying ß-arrestin-biased signaling-pathway-mediated ERK activation. We found that the stimulation of the D2R ß-arrestin signaling pathway caused Mdm2, an E3 ubiquitin ligase, to move from the nucleus to the cytoplasm and interact with tyrosine phosphorylated G-protein-coupled receptor kinase 2 (GRK2), which was facilitated by Src, a non-receptor tyrosine kinase. This interaction led to the ubiquitination of GRK2, which then moved to the plasma membrane and interacted with activated D2R, followed by the phosphorylation of D2R as well as the mediation of ERK activation. In conclusion, Mdm2-mediated GRK2 ubiquitination, which is selectively triggered by the stimulation of the D2R ß-arrestin signaling pathway, is necessary for GRK2 membrane translocation and its interaction with D2R, which in turn mediates downstream ERK signaling. This study is primarily novel and provides essential information with which to better understand the detailed mechanisms of D2R-dependent signaling.

Biphasic transcriptional and posttranscriptional regulation of MYB by androgen signaling mediates its growth control in prostate cancer.

MYB, a proto-oncogene, is overexpressed in prostate cancer (PCa) and promotes its growth, aggressiveness, and resistance to androgen-deprivation therapy. Here, we examined the effect of androgen signaling on MYB expression and delineated the underlying molecular mechanisms. Paralleling a dichotomous effect on growth, low-dose androgen induced MYB expression at both transcript and protein levels, whereas it was suppressed in high-dose androgen-treated PCa cells. Interestingly, treatment with both low- and high-dose androgen transcriptionally upregulated MYB by increasing the binding of androgen receptor to the MYB promoter. In a time-course assay, androgen induced MYB expression at early time points followed by a sharp decline in high-dose androgen-treated cells due to decreased stability of MYB mRNA. Additionally, profiling of MYB-targeted miRNAs demonstrated significant induction of miR-150 in high-dose androgen-treated PCa cells. We observed a differential binding of androgen receptor on miR-150 promoter with significantly greater occupancy recorded in high-dose androgen-treated cells than those treated with low-dose androgen. Functional inhibition of miR-150 relieved MYB suppression by high-dose androgen, while miR-150 mimic abolished MYB induction by low-dose androgen. Furthermore, MYB-silencing or miR-150 mimic transfection suppressed PCa cell growth induced by low-dose androgen, whereas miR-150 inhibition rescued PCa cells from growth repression by high-dose androgen. Similarly, we observed that MYB silencing suppressed the expression of androgen-responsive, cell cycle-related genes in low-dose androgen-treated cells, while miR-150 inhibition increased their expression in cells treated with high-dose androgen. Overall, these findings reveal novel androgen-mediated mechanisms of MYB regulation that support its biphasic growth control in PCa cells.

Anisotropy of Additively Manufactured Co-28Cr-6Mo Influences Mechanical Properties and Biomedical Performance.

Additive manufacturing (AM) of biomedical alloys such as Co-Cr-Mo alloys holds immense potential for fabricating implants with complex geometry and tailored to meet patient-specific needs. However, layer-by-layer fabrication in AM processes results in undesired anisotropy due to the solidification texture and grain morphology. The present study aimed to investigate the effect of build orientation on the mechanical properties and functional performance, including tribocorrosion behavior and cytocompatibility of an orthopedic Co-28Cr-6Mo alloy manufactured by selective laser melting. Although the fabricated alloy showed weak crystallographic texture due to the rotational scanning strategy, significant anisotropy was found in the tensile properties due to the grain size and morphology. The presence of larger, elongated grains along the build direction as compared to smaller, equiaxed grains perpendicular to the build direction imparted the observed tensile anisotropy. Quantitative analysis based on current models for strengthening mechanisms is insufficient to explain the observed anisotropy, which is ascribed to the possible role of the cellular dendrites and stacking fault strengthening in Co-Cr alloys. Unlike the electrochemical behavior, which was largely independent of the build orientation, the bio-tribocorrosion studies revealed an anisotropic wear rate under fretting conditions. Osteoblast attachment and proliferation were found to be higher on the plane perpendicular to the build direction, owing to the differences in grain size. This work provides novel insights into the role of the manufacturing parameters in a selective-laser-melted Co-Cr alloy and its potential application in engineering load-bearing orthopedic implants.

Nicotine causes alternative polarization of macrophages via Src-mediated STAT3 activation: Potential pathobiological implications.

Nicotine is an addictive ingredient of tobacco products and other noncigarette substitutes, including those being used for smoking cessation to relieve withdrawal symptoms. Earlier research, however, has associated nicotine with the risk and poorer outcome of several diseases, including cancer. Macrophages are an important component of the innate immune system and can have both pro-and anti-inflammatory functions depending upon their polarization state. Here, we investigated the effect of nicotine on macrophage polarization, growth, and invasion to understand its role in human physiology. We observed that nicotine induced M2 polarization of RAW264.7 and THP-1-derived macrophages in a dose-dependent manner. Cytokine profiling suggested a mixed M2a/d phenotype of nicotine-polarized macrophages associated with tissue repair and pro-angiogenic functions. Moreover, nicotine treatment also enhanced the growth, motility, and invasion of macrophages. Mechanistic studies revealed increased phosphorylation of STAT3 in nicotine-treated macrophages that was mediated through Src activation. Importantly, pretreatment of macrophages with either Src or STAT3 inhibitor abrogated nicotine-induced macrophage polarization, growth, and motility, suggesting a functional role of the Src-STAT3 signaling axis. Together, our findings reveal a novel role of nicotine in immunosuppression via causing M2 polarization of macrophages that could be implicated in the pathogenesis of various diseases.

Clinicopathologic significance and race-specific prognostic association of MYB overexpression in ovarian cancer.

Late diagnosis, unreliable prognostic assessment, and poorly-guided therapeutic planning result in dismal survival of ovarian cancer (OC) patients. Therefore, identifying novel functional biomarker(s) is highly desired for improved clinical management. MYB is an oncogenic transcription factor with emerging functional significance in OC. Here we examined its clinicopathologic significance by immunohistochemistry and TCGA/GTex data analyses. Aberrant MYB expression was detected in 94% of OC cases (n = 373), but not in the normal ovarian tissues (n = 23). MYB was overexpressed in all major epithelial OC histological subtypes exhibiting the highest incidence (~ 97%) and overall expression in serous and mucinous carcinomas. MYB expression correlated positively with tumor grades and stages. Moreover, MYB exhibited race-specific prognostic association. Moderate-to-high MYB levels were significantly associated with both poor overall- (p = 0.02) and progression-free (p = 0.02) survival in African American (AA), but not in the Caucasian American (CA) patients. Consistent with immunohistochemistry data, we observed significantly higher MYB transcripts in OC cases (n = 426) than normal ovary (n = 88). MYB transcripts were significantly higher in all epithelial OC subtypes, compared to normal, and its greater levels predicted poor survival in AA OC, but not CA OC, patients. Thus, MYB appears to be a useful clinical biomarker for prognostication, especially in AA patients.

Structural Requirements for Modulating 4-Benzylpiperidine Carboxamides from Serotonin/Norepinephrine Reuptake Inhibitors to Triple Reuptake Inhibitors.

8K: and a serotonin/norepinephrine reuptake inhibitor. 7J: showed that the regions spanning transmembrane domain (TM)1, TM3, and TM6 form the ligand binding pocket. The compound. 8K: bound tightly to the binding pocket of all three monoamine reuptake transporters; however. 7J: showed poor docking with DAT. Co-expression of DAT with the dopamine D2 receptor (D2R) significantly inhibited DA-induced endocytosis of D2R probably by reuptaking DA into the cells. Pretreatment of the cells with. 8F: , which is one of the compounds with good inhibitory activity on DAT, blocked DAT-induced inhibition of D2R endocytosis. In summary, this study identified critical structural features contributing to the selectivity of a molecule for each of the monoamine transporters, critical residues on the compounds that bound to the transporters, and the functional role of a DA reuptake inhibitor in regulating D2R function.

Zinc and cerium synergistically enhance the mechanical properties, corrosion resistance, and osteogenic activity of magnesium as resorbable biomaterials.

Magnesium and its alloys have the potential to serve as a revolutionary class of biodegradable materials, specifically in the field of degradable implants for orthopedics. However, the corrosion rate of commercially pure magnesium is high and does not match the rate of regeneration of bone tissues. In this work, magnesium alloys containing zinc and cerium, either alone or in combination, were investigated and compared with commercially-pure magnesium as biomaterials. The microstructure, mechanical properties, corrosion resistance, and response of osteoblastsin vitrowere systematically assessed. Results reveal that alloying with Ce results in grain refinement and weakening of texture. The tensile test revealed that the ternary alloy offered the best combination of elastic modulus (41.1 ± 0.5 GPa), tensile strength (234.5 ± 4.5 MPa), and elongation to break (17.1 ± 0.4%). The ternary alloy was also the most resistant to corrosion (current of 0.85 ± 0.05 × 10-4A cm-2) in simulated body fluid than the other alloys. The response of MC3T3-E1 cellsin vitrorevealed that the ternary alloy imparts minimal cytotoxicity. Interestingly, the ternary alloy was highly efficient in supporting osteogenic differentiation, as revealed by the expression of alkaline phosphatase and calcium deposition. In summary, the extruded Mg alloy containing both Zn and Ce exhibits a combination of mechanical properties, corrosion resistance, and cell response that is highly attractive for engineering biodegradable orthopedic implants.

R (-)-methoxetamine exerts rapid and sustained antidepressant effects and fewer behavioral side effects relative to S (+)-methoxetamine.

The newfound antidepressant efficacy of ketamine has provided opportunities for the development of new-generation, rapid-acting, glutamate-based antidepressants. We previously identified that methoxetamine (MXE), a ketamine analog, and an N-Methyl-d-aspartate (NMDA) receptor antagonist, produced rapid and sustained antidepressant effects in mice. MXE (R, S (±)-MXE) is a racemic mixture containing equal parts of S (+)-MXE and R (-)-MXE. However, studies have yet to investigate the antidepressant effects of its enantiomers. Here, we examined the potential antidepressant properties and behavioral side effects of S- and R-MXE in mice. Both S- and R-MXE showed significant NMDA receptor affinity and appreciable inhibitory activity on serotonin transporter. Also, S- and R-MXE (10 mg kg-1) exerted antidepressant effects and increased gamma waves (electroencephalography) but were inhibited by NBQX (an AMPA receptor antagonist). Subsequently, they increased mammalian target of rapamycin phosphorylation and AMPA receptor subunits GluA1 and GluA2 protein levels in the hippocampus or prefrontal cortex. Furthermore, they increased 5HT2a and 5HT2c receptor mRNA levels in the prefrontal cortex, with their antidepressant effects inhibited by ketanserin (a 5HT2a/c receptor antagonist). Taken together, S-MXE and R-MXE elicit antidepressant effects that are probably mediated via glutamatergic and serotonergic mechanisms. Unlike S-MXE, R-MXE did not induce prepulse inhibition deficits, hyperlocomotion, conditioned place preference, and locomotor sensitization, although it acutely altered motor coordination. This suggests that R-MXE induces fewer behavioral side effects and is a safer antidepressant than S-MXE. Overall, this study provides significant implications for future research on the next generation of rapid-acting, glutamate-based antidepressant drugs.

Roles of the Functional Interaction between Brain Cholinergic and Dopaminergic Systems in the Pathogenesis and Treatment of Schizophrenia and Parkinson's Disease.

Most physiologic processes in the brain and related diseases involve more than one neurotransmitter system. Thus, elucidation of the interaction between different neurotransmitter systems could allow for better therapeutic approaches to the treatments of related diseases. Dopaminergic (DAergic) and cholinergic neurotransmitter system regulate various brain functions that include cognition, movement, emotion, etc. This review focuses on the interaction between the brain DAergic and cholinergic systems with respect to the pathogenesis and treatment of schizophrenia and Parkinson's disease (PD). We first discussed the selection of motor plans at the level of basal ganglia, the major DAergic and cholinergic pathways in the brain, and the receptor subtypes involved in the interaction between the two signaling systems. Next, the roles of each signaling system were discussed in the context of the negative symptoms of schizophrenia, with a focus on the α7 nicotinic cholinergic receptor and the dopamine D1 receptor in the prefrontal cortex. In addition, the roles of the nicotinic and dopamine receptors were discussed in the context of regulation of striatal cholinergic interneurons, which play crucial roles in the degeneration of nigrostriatal DAergic neurons and the development of L-DOPA-induced dyskinesia in PD patients. Finally, we discussed the general mechanisms of nicotine-induced protection of DAergic neurons.

ß-Arrestin1 and GPCR kinase2 play permissive roles in Src-mediated endocytosis of α4ß2 nicotinic ACh receptors.

BACKGROUND AND PURPOSE: The α4ß2 nicotinic ACh receptor (nAChR), a subtype of the ligand-gated ion channel, is abundantly expressed in the brain and is implicated in several neurological disorders. The endocytosis of nAChRs plays important roles in the pathogenesis of neurological diseases, but the underlying molecular mechanisms remain poorly understood. EXPERIMENTAL APPROACH: Loss-of-function approaches and mutants of α4ß2 nAChRs that display different endocytic properties were used to identify the cellular components and processes responsible for endocytosis. The signalling cascade that leads to endocytosis was deduced via protein interactions in predicted cellular components. The endocytosis of α4ß2 nAChRs was determined and crosschecked using an ELISA and radioligand assay. KEY RESULTS: Endocytosis of α4ß2 nAChRs occurred through clathrin-mediated endocytosis in a dynamin-dependent manner. 14-3-3η-dependent Src-mediated phosphorylation of the nAChR α4 subunit at Y575 was required for nAChR endocytosis, and this occurred with the assistance of ß-arrestin1 and GPCR kinase 2 (GRK2) without the need for kinase activity. Endocytosis triggered the mouse double minute 2 homologue-mediated ubiquitination and subsequent down-regulation of α4ß2 nAChRs. CONCLUSIONS AND IMPLICATIONS: α4ß2 nAChR, an ionophore receptor, employs the metabotropic signalling pathway required for endocytosis, which leads to ubiquitination and down-regulation. Further, GRK2 and ß-arrestin1, usually associated with GPCR signalling, are involved in the endocytosis of α4ß2 nAChRs via different mechanisms. Considering the functional and pathological implications of nAChR endocytosis, results obtained in this study are crucial for the progression of basic research and clinical investigations.

Review of recent developments in surface nanocrystallization of metallic biomaterials.

Metallic materials are widely used to prepare implants for both short-term and long-term use in the human body. The performance of these implants is greatly influenced by their surface characteristics, which has motivated the development of several surface modification techniques. Surface severe plastic deformation (S2PD) techniques have emerged as promising strategies to enhance the performance of metallic biomaterials. They do not involve chemical modification of the surface and impart minimal changes to the surface topography. S2PD processes are based on the principle of generating nanocrystals at the surface, which can improve performance metrics, such as fatigue, wear, corrosion resistance, and biocompatibility through various mechanisms, such as surface hardening and alterations to the surface oxide layer. This review presents the state of the art on the development of different S2PD processes and their applications on metallic biomaterials. Brief descriptions of the different processes have been provided, followed by a discussion on the microstructural changes induced by these processes for different generations of biomaterials. The effect of S2PD on surface and bulk characteristics of the biomaterials and their performance is critically reviewed. As an emerging class of surface engineering techniques in biomaterials science, more work is needed to fully leverage their potential in this field, and these opportunities are discussed in this review.

A novel molecular mechanism responsible for phosphorylation-independent desensitization of G protein-coupled receptors exemplified by the dopamine D3 receptor.

GRK-mediated receptor phosphorylation followed by association with ß-arrestins has been proposed to be the molecular mechanism involved in the desensitization of G protein-coupled receptors (GPCRs). However, this mechanism does not explain the desensitization of some GPCRs, such as dopamine D3 receptor (D3R), which does not undergo GRK-mediated phosphorylation. Loss-of-function approaches and mutants of dopamine D2 receptor and D3R, which exhibit different desensitization properties, were used to identify the cellular components and processes responsible for desensitization. D3R mediated the recruitment of Mdm2 to the cytosol, which resulted in the constitutive ubiquitination of ß-arrestin2 in the resting state. Under desensitization conditions, cytosolic Mdm2 returned to the nucleus, resulting in the deubiquitination of cytosolic ß-arrestins. Deubiquitinated ß-arrestins formed a tight complex with Gßγ, thereby sequestering it, causing interference in D3R signaling. In conclusion, this study shows that ß-arrestins, depending on their ubiquitination status, control the G protein cycling by regulating their interactions with Gßγ. This is a novel mechanism proposed to explain how certain GPCRs can undergo desensitization without receptor phosphorylation.

Metabotropic signaling cascade involved in α4ß2 nicotinic acetylcholine receptor-mediated PKCßII activation.

Nicotinic acetylcholine receptors (nAChRs) belong to the ionophore receptor family, which regulates plasma membrane conductance to Na+, K+, and Ca2+ ions. Some studies, however, have shown that nAChRs also employ second messengers for intracellular signaling. We previously showed that α4ß2 nAChR mediates the translocation of protein kinase CßII (PKCßII) from the cytoplasm to the plasma membrane, which is a typical activation marker for PKCßII. In this study, we investigated the molecular mechanisms underlying PKCßII activation through α4ß2 nAChR. α4ß2 nAChR is the most abundant nAChR subtype and is implicated in various brain functions and diseases. Putative α4ß2 nAChR signaling components were identified by knockdown or chemical inhibition of candidate proteins, and the signaling cascade was deduced by protein interactions in predicted cellular components. α4ß2 nAChR-mediated PKCßII translocation was found to occur in an ionophore activity-independent manner. Nicotinic stimulation of α4ß2 nAChR activated Src in a ß-arrestin1 and 14-3-3η-dependent manner. Activated Src phosphorylated the tyrosine residue(s) on Syk molecules, which in turn interacted with phospholipase C γ1 to trigger the translocation of PKCßII to the cell membrane by elevating cellular diacylglycerol levels. The activated PKCßII in turn exerted a positive feedback effect on Src activation, suggesting that α4ß2 nAChR signaling is amplified by a positive feedback loop. These findings provide novel information for unveiling the previously unclear metabotropic second messenger-based signal transduction pathway of nAChRs.

Surface mechanical attrition treatment of low modulus Ti-Nb-Ta-O alloy for orthopedic applications.

Surface mechanical attrition treatment (SMAT) is recognized as a surface severe plastic deformation (SPD) method that is effective in improving the surface-dependent mechanical and functional properties of conventional metallic biomaterials. In this study, we aimed to systemically investigate the effect of SMAT on the physical, electrochemical, tribological and biological performances of a newly developed low modulus ß Ti-Nb-Ta-O alloy with two different microstructures, namely, single phase ß-treated and dual phase ß + α aged. The microhardness results showed considerable hardening for the ß-treated condition due to formation of deformation substructures; that was associated with increased corrosion resistance resulting from a stronger and denser passive layer on the surface, as revealed by Tafel polarization, impedance studies and Mott-Scottky plots. The wear volume loss during fretting in serum solution was found to decrease by 46% while friction coefficient decreased only marginally, due to presence of a harder and more brittle surface. In the ß + α condition of the alloy, minimal hardening was observed due to coarsening of the precipitates during SMAT. However, this also reduced the number of α-ß interfaces, which in turn minimized the tendency for galvanic corrosion resulting in lower corrosion rate after SMAT. Wear resistance was enhanced after SMAT, with 32% decrease in wear volume loss and 21% decrease in friction coefficient resulted due to improved ductility on the surface. The attachment and growth of osteoblasts on the alloys in vitro were not affected by SMAT and was comparable to that on commercially pure Ti. Taken together, these results provide new insights into the effects of surface SPD of low modulus ß- Ti alloys for orthopedic applications and underscore the importance of the initial microstructure in determining the performance of the alloy.

Catalpol and Mannitol, Two Components of Rehmannia glutinosa, Exhibit Anticonvulsant Effects Probably via GABAA Receptor Regulation.

Epilepsy is a brain disorder that affects millions of people worldwide and is usually managed using currently available antiepileptic drugs, which result in adverse effects and are ineffective in approximately 20-25% of patients. Thus, there is growing interest in the development of new antiepileptic drugs with fewer side effects. In a previous study, we showed that a Rehmannia glutinosa (RG) water extract has protective effects against electroshock- and pentylenetetrazol (PTZ)-induced seizures, with fewer side effects. In this study, the objective was to identify the RG components that are responsible for its anticonvulsant effects. Initially, a number of RG components (aucubin, acteoside, catalpol, and mannitol) were screened, and the anticonvulsant effects of different doses of catalpol, mannitol, and their combination on electroshock- and chemically (PTZ or strychnine)-induced seizures in mice, were further assessed. Gamma-aminobutyric acid (GABA) receptor binding assay and electroencephalography (EEG) analysis were conducted to identify the potential underlying drug mechanism. Additionally, treated mice were tested using open-field and rotarod tests. Catalpol, mannitol, and their combination increased threshold against electroshock-induced seizures, and decreased the percentage of seizure responses induced by PTZ, a GABA antagonist. GABA receptor binding assay results revealed that catalpol and mannitol are associated with GABA receptor activity, and EEG analysis provided evidence that catalpol and mannitol have anticonvulsant effects against PTZ-induced seizures. In summary, our results indicate that catalpol and mannitol have anticonvulsant properties, and may mediate the protective effects of RG against seizures.

Mdm2-mediated ubiquitination of PKCßII in the nucleus mediates clathrin-mediated endocytic activity.

Cellular stimuli that increase diacylglycerol levels activate several protein kinase C (PKC) isoforms; however, prolonged stimulation depletes cells of PKCs. Ubiquitination is a critical cellular event that mediates the degradation of numerous proteins, including PKCs, but little is known of the molecular mechanisms involved in PKC ubiquitination. PKCßII is the most widely expressed PKC isoform and regulates a variety of cellular functions. Here, we show that in response to stimulation of the Gq-coupled angiotensin II type 1 receptor or treatment with phorbol ester, Mdm2, E3 ubiquitin ligase, interacted with PKCßII isotype in the nucleus, resulting in ubiquitination of PKCßII at the C-terminal K668 and K672 residues and its subsequent downregulation. Ubiquitinated PKCßII mediated the clathrin-mediated endocytosis of G protein-coupled receptors like the D2 and D3 dopamine receptors; in contrast, non-ubiquitinated PKCßII mediated an as yet uncharacterized clathrin- and caveolar-independent endocytic pathway. In conclusion, we characterized the molecular mechanisms involved in the activity-dependent ubiquitination of PKCßII that determine its life span and endocytic roles. Considering that PKCßII plays an important role in the development of various diseases, including diabetic vasculitis, the results obtained in this study will contribute to better understanding the pathogenesis of PKCßII-related diseases.