Intracellular Molecular Mechanism Research Articles

ApoM maintains cellular homeostasis between mitophagy and apoptosis by affecting the stability of Nnt mRNA through the Zic3-ApoM-Elavl2-Nnt axis during neural tube closure

Research on the aetiology of neural tube defects (NTDs) has made progress in recent years. However, the molecular mechanism of apolipoproteins underlying NTDs development remains unclear. This study aimed to investigate the function of apolipoprotein M (ApoM) in the pathogenesis of NTDs and its underlying mechanisms. We demonstrated that ApoM expression was reduced in the spinal cord samples of rat models and human fetuses with NTDs respectively. Specifically, lack of ApoM resulted in reduced cytosolic localization of Elavl2 and caused Nnt mRNA degradation, which further led to impaired cell homeostasis by suppressing PINK1-PRKN-mediated mitophagy and promoting apoptosis and subsequent NTDs formation. Moreover, Zic3 directly interacted with the promoter of ApoM and activated its transcription. Lastly, intra-amniotic delivery of adenoviral recombinant Zic3 or ApoM could promote mitophagy and alleviate apoptosis in spinal cords of NTDs. Collectively, these findings highlight the important role of the Zic3-ApoM-Elavl2-Nnt axis in cellular homeostasis during neural tube development, thereby revealing an intracellular molecular regulatory mechanism of ApoM, providing a mechanistic basis for understanding embryonic neural development, and offering experimental evidence for potential therapeutic targets for NTDs.

Bioimaging and Sensing Thiols In Vivo and in Tumor Tissues Based on a Near-Infrared Fluorescent Probe with Large Stokes Shift.

The well-known small-molecule biothiols have been used to maintain the normal metabolism of peroxy radicals, forming protein structures, resisting cell apoptosis, regulating metabolism, and protecting the homeostasis of cells in the organism. A large amount of research has found that abnormal levels of the above biothiols can cause some adverse diseases, such as changes in hair pigmentation, a slower growth rate, delayed response, excessive sleep and skin diseases. In order to further investigate the exact intracellular molecular mechanism of biothiols, it is imperative to explore effective strategies for real-time biothiol detection in living systems. In this work, a new near-infrared (NIR) emission fluorescence probe (probe 1) for sensitive and selective detection of biothiols was devised by combining dicyanoisophorone derivatives with the dinitrobenzenesulfonyl (DNBS) group. As expected, probe 1 could specifically detect biothiols (Cys, Hcy and GSH) through the dinitrobenzenesulfonyl group to form dye 2, which works as a signaling molecule for sensing biothiols in real samples. Surprisingly, probe 1 showed superior sensing characteristics and low-limit detection towards biothiols (36.0 nM for Cys, 39.0 nM for Hcy and 48.0 nM for GSH) with a large Stokes shift (134 nm). Additionally, the function of probe 1 as a platform for detecting biothiols was confirmed by confocal fluorescence imaging of biothiols in MCF-7 cells and zebrafish. More importantly, the capability of probe 1 in vivo has been further evaluated by imaging the overexpressed biothiols in tumor tissue. It is reasonable to believe that probe 1 can provide a valuable method to explore the relationship between biothiols and the genesis of tumor.

The therapeutic effects of low-intensity pulsed ultrasound in musculoskeletal soft tissue injuries: Focusing on the molecular mechanism.

Musculoskeletal soft tissue injuries are very common and usually occur during both sporting and everyday activities. The intervention of adjuvant therapies to promote tissue regeneration is of great importance to improving people's quality of life and extending their productive lives. Though many studies have focused on the positive results and effectiveness of the LIPUS on soft tissue, the molecular mechanisms standing behind LIPUS effects are much less explored and reported, especially the intracellular signaling pathways. We incorporated all research on LIPUS in soft tissue diseases since 2005 and summarized studies that uncovered the intracellular molecular mechanism. This review will also provide the latest evidence-based research progress in this field and suggest research directions for future experiments.

GCRV NS38 counteracts SVCV proliferation by intracellular antagonization during co-infection

Viral co-infection has been found in animals; however, the mechanisms of co-infection are unclear. The abundance and diversity of viruses in water make fish highly susceptible to co-infection. Here, we reported a co-infection in fish, which resulted in reduced host lethality and illustrated the intracellular molecular mechanism of viral co-infection. The spring viremia of carp virus (SVCV) is a highly lethal virus that infects Cyprinidae, such as zebrafish. The mortality of SVCV infection was significantly reduced when co-infected with the grass carp reovirus (GCRV). The severity of tissue damage and viral proliferation of SVCV was also reduced in co-infection with GCRV. The transcriptome bioinformatics analysis demonstrated that the effect on the host transcripts in response to SVCV infection was significantly reduced in co-infection. After excluding the extracellular interactions of these two viruses, the intracellular mechanisms were studied. We found that the GCRV NS38 remarkably decreased SVCV infection and viral proliferation. The interaction between GCRV NS38 and SVCV nucleoprotein (N) and phosphoprotein (P) proteins was identified, and NS38 downregulated both N and P proteins. Further analysis demonstrated that the N protein was degraded by NS38 indispensable of the autophagy receptor, sequestosome 1 (p62). Meanwhile, K63-linked ubiquitination of the P protein was reduced by NS38, leading to ubiquitinated degradation of the P protein. These results reveal that the intracellular viral protein interactions are a crucial mechanism of co-infection and influence the host pathology and expand our understanding in intracellular viral interactions co-infection.

Identification of miRNA signature associated with BMP2 and chemosensitivity of TMZ in glioblastoma stem-like cells

Glioblastoma multiform (GBM) is the most lethal intracranial tumor in adults. Glioblastoma stem-like cells (GSCs) are responsible for tumorigenesis and chemotherapy resistance. BMPs are known to increase temozolomide (TMZ) response in GSCs, however, the intracellular molecular mechanism remains largely unknown. In this study, we built a GSC cell model called U87S, and performed RNA sequencing to identify differentially expressed (DE) miRNA profiles in U87S cells treated with BMP2, TMZ or combined BMP2 and TMZ respectively. Bioinformatics analysis revealed that most DE miRNAs were involved in the cancer pathways, suggesting their crucial roles in gliomagenesis. Eight miRNAs from RNA-seq were validated. Four out of these miRNAs (has-miR-199a-3p, hsa-miR-374b-5p, hsa-miR-320d, and hsa-miR-339-5p) were found significantly up-regulated in GBM tumor tissues. One of them, hsa-miR-199a-3p, was significantly correlated with the survival of GBM patients, and differentially expressed in U87S cells. Expression of hsa-miR-199a-3p was up-regulated by BMP. Overexpression of hsa-miR-199a-3p in U87S cells inhibited cell viability and enhanced the cytotoxicity of TMZ. And activation of BMP boosted the effect of hsa-miR-199a-3p on cell viability and TMZ-mediated cytotoxicity. Besides, expressions of five predicted targets of hsa-miR-199a-3p were evaluated. Four of them were differentially expressed in GBM tumors. And one of them, SLC22A18, was associated with the survival of GBM patients. In the end, a hsa-miR-199a-3p-mediated ceRNA network was constructed for the convenience of future study. Together, our data provided DE miRNA expression profiles associated with BMP2 and TMZ in GSCs, which might lead to finding out miRNA-based target therapies that specially target GSCs.

Downregulation of AKT and MDM2, Melatonin Induces Apoptosis in AGS and MGC803 Cells.

Melatonin, a neurohormone secreted by the pineal gland, has a variety of biological functions, such as circadian rhythms regulation, anti-oxidative activity, immunomodulatory effects, and anittumor, etc. At present, its antitumor effect has attracted people's attention due to its extensive tissue distribution, good tissue compatibility, and low toxic and side effects. In the gastrointestinal tract, there is high level of melatonin and many studies showed melatonin has effects of anti-gastric cancer. In this experiment, human gastric cancer cell lines AGS and MGC803 were used to investigate the intracellular molecular mechanism of melatonin against gastric cancer. After AGS and MGC803 have been treated with melatonin, the changes of cell morphology and cellular structure were observed under electron microscope. Flow cytometer and apoptosis detection kits were used to analyze the effect of apoptosis on AGS and MGC803. The alterations of apoptosis-related proteins Caspase 9, Caspase 3, and upstream regulators AKT, MDM2 including expression, phosphorylation, and activation were detected to analyze the intracellular molecular mechanism of melatonin inhibiting gastric cancer. In AGS and MGC803 cells with melatonin exposure, cleaved Caspase 9 was upregulated and Caspase 3 was activated; moreover, MDM2 and AKT expression and phosphorylation were downregulated. Melatonin promoted apoptosis of AGS and MGC803 cells by the downregulation of AKT and MDM2. Anat Rec, 302:1544-1551, 2019. © 2019 American Association for Anatomy.

Noradrenergic β-Adrenoceptor-Mediated Intracellular Molecular Mechanism of Na-K ATPase Subunit Expression in C6 Cells.

Rapid eye movement sleep deprivation-associated elevated noradrenaline increases and decreases neuronal and glial Na-K ATPase activity, respectively. In this study, using C6 cell-line as a model, we investigated the possible intracellular molecular mechanism of noradrenaline-induced decreased glial Na-K ATPase activity. The cells were treated with noradrenaline in the presence or absence of adrenoceptor antagonists, modulators of extra- and intracellular Ca++ and modulators of intracellular signalling pathways. We observed that noradrenaline acting on β-adrenoceptor decreased Na-K ATPase activity and mRNA expression of the catalytic α2-Na-K ATPase subunit in the C6 cells. Further, cAMP and protein kinase-A mediated release of intracellular Ca++ played a critical role in such decreased α2-Na-K ATPase expression. In contrast, noradrenaline acting on β-adrenoceptor up-regulated the expression of regulatory β2-Na-K ATPase subunit, which although was cAMP and Ca++ dependent, was independent of protein kinase-A and protein kinase-C. Combining these with previous findings (including ours) we have proposed a working model for noradrenaline-induced suppression of glial Na-K ATPase activity and alteration in its subunit expression. The findings help understanding noradrenaline-associated maintenance of brain excitability during health and altered states, particularly in relation to rapid eye movement sleep and its deprivation when the noradrenaline level is naturally altered.

Defect-Related Luminescent Hydroxyapatite-Enhanced Osteogenic Differentiation of Bone Mesenchymal Stem Cells Via an ATP-Induced cAMP/PKA Pathway.

Novel defect-related hydroxyapatite (DHAP), which combines the advantages of HAP and defect-related luminescence, has the potential application in tissue engineering and biomedical area, because of its excellent capability of monitoring the osteogenic differentiation and material biodegradation. Although the extracellular mechanism of DHAP minerals and PO4(3-) functioning in osteogenic differentiation has been widely studied, the intracellular molecular mechanism through which PO4(3-) mediates osteogenesis of bone mesenchymal stem cells (BMSCs) is not clear. We examined a previously unknown molecular mechanism through which PO4(3-) promoted osteogenesis of BMSCs with an emphasis on adenosine-triphosphate (ATP)-induced cAMP/PKA pathway. Our studies showed that DHAP could be uptaken into lysosome, in which PO4(3-) was released from DHAP, because of the acid environment of lysosome. The released PO4(3-) interacted with ADP to form ATP, and then degraded into adenosine, an ATP metabolite, which interacted with A2b adenosine receptor to activate the cAMP/PKA pathway, resulting in the high expression of osteogenesis-related genes, such as Runx2, BMP-2, and OCN. These findings first revealed the function of ATP-metabolism in bone physiological homeostasis, which may be developed to cure bone metabolic diseases.

Arf6 guanine-nucleotide exchange factor cytohesin-2 regulates myelination in nerves

In postnatal development of the peripheral nervous system (PNS), Schwann cells differentiate to insulate neuronal axons with myelin sheaths, increasing the nerve conduction velocity. To produce the mature myelin sheath with its multiple layers, Schwann cells undergo dynamic morphological changes. While extracellular molecules such as growth factors and cell adhesion ligands are known to regulate the myelination process, the intracellular molecular mechanism underlying myelination remains unclear. In this study, we have produced Schwann cell-specific conditional knockout mice for cytohesin-2, a guanine-nucleotide exchange factor (GEF) specifically activating Arf6. Arf6, a member of the Ras-like protein family, participates in various cellular functions including cell morphological changes. Cytohesin-2 knockout mice exhibit decreased Arf6 activity and reduced myelin thickness in the sciatic nerves, with decreased expression levels of myelin protein zero (MPZ), the major myelin marker protein. These results are consistent with those of experiments in which Schwann cell-neuronal cultures were treated with pan-cytohesin inhibitor SecinH3. On the other hand, the numbers of Ki67-positive cells in knockout mice and controls are comparable, indicating that cytohesin-2 does not have a positive effect on cell numbers. Thus, signaling through cytohesin-2 is required for myelination by Schwann cells, and cytohesin-2 is added to the list of molecules known to underlie PNS myelination.

PTEN plays an important role in thrombin-mediated lung cancer cell functions.

Thrombin and its membrane receptor, protease-activated receptor 1 (PAR1), have been reported to promote the development of lung cancer in vitro and in vivo. However, the intracellular molecular mechanism or signaling pathway that mediates the cytological effects after the thrombin-receptor interaction is poorly understood. Our previous study observed that the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was downregulated in thrombin-stimulated lung cancer. In this study, the role of PTEN in thrombin-mediated cell function and the corresponding cell signaling pathway were studied in lung cancer cell Glc-82. The results indicated that thrombin downregulates the PTEN expression level and that PTEN plays an important role in thrombin-mediated Glc-82 functions, including cell cycle progression, cell apoptosis, and cell migration. The PI3K/AKT signaling pathway and its related proteins, including p27 and S phase kinase associated protein 2 (Skp2), are involved in the effects induced by PTEN downregulation. PAR1 plays a role in thrombin-mediated reduction of PTEN expression. This study suggested that the PTEN/PI3K/AKT signaling pathway plays an important role in thrombin/PAR1-mediated lung cancer cell growth and migration.

Plasma Medical Science for Cancer Therapy: Toward Cancer Therapy Using Nonthermal Atmospheric Pressure Plasma

We have been developing novel ultrahigh density atmospheric pressure plasma sources and succeeded in the selective killing ovarian cancer cells against normal ones. Furthermore, we have found out the plasma-activated medium (PAM) also killed glioblastoma brain tumor cells selectively against normal ones and the chemical products in the PAM have long lifetime healing effects. To clarify the mechanism, interactions of plasma with the organism and the medium where the organism belongs were investigated on the viewpoint of intracellular molecular mechanism.

Pelizaeus–Merzbacher disease-associated proteolipid protein 1 inhibits oligodendrocyte precursor cell differentiation via extracellular-signal regulated kinase signaling

Oligodendrocytes (OLs) are myelin-forming glial cells in the central nervous system (CNS) and their dysfunction causes neuropathies such as demyelinating diseases. Proteolipid protein 1 (PLP1) is an oligodendrocyte myelin-rich tetraspan membrane protein and aberration of the plp1 gene is known to be responsible for dysmyelinating Pelizaeus–Merzbacher disease (PMD). Among previously identified gene alternations, multiplication of the plp1 gene causes increased expression of PLP1, resulting in a phenotype with severe dysmyelination in human and also rodent models. Yet little is known about the relationship between increased PLP1 expression and oligodendrocyte precursor cell (OPC) differentiation and the intracellular molecular mechanism. Here we show that expression of PLP1 in OPCs markedly inhibits their differentiation, and that this inhibitory effect is effectively improved by inhibition of extracellular signal-regulated kinase (ERK) activity. Furthermore, in cocultures with dorsal root ganglion (DRG) neurons, ERK inhibition also improves PLP1-induced dysmyelination. Thus, ERK inhibition helps to improve defective OPC differentiation induced by PLP1 expression, suggesting that molecules belonging to ERK signaling cascade may be new PMD therapeutic targets.

Thymic stromal lymphopoietin promotes the proliferation of human trophoblasts via phosphorylated STAT3-mediated c-Myc upregulation

Our previous study has demonstrated that thymic stromal lymphopoietin (TSLP) stimulates trophoblast proliferation and invasion, suggesting TSLP plays an important role in the placentation in early human pregnancy, but the intracellular molecular mechanism is currently unknown. The present study is undertaken to investigate whether the STAT3-c-Myc signaling pathway is involved in TSLP-mediated trophoblast proliferation. Primary human first-trimester trophoblasts were treated with TSLP only, or TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, and ERK). The levels of STAT3 tyrosine phosphorylation and c-Myc expression were determined by using Western blot analysis, and the proliferation of trophoblasts was analyzed by BrdU cell proliferation assay. JEG-3 cells were transfected with siRNA targeting to c-Myc, and the proliferation was determined in JEG-3 cells treated with TSLP only, or TSLP combined with c-Myc silencing. It was revealed that treatment with TSLP significantly enhanced STAT3 phosphorylation and c-Myc expression in human trophoblasts. The effect of TSLP upregulation on trophoblast proliferation was abrogated completely by either STAT3 inhibitor or c-Myc siRNA silence. We further found that the upregulation of TSLP on c-Myc expression was abrogated completely by the STAT3 inhibitor, which suggests that the intracellular STAT3 phosphorylation is an upstream signal of c-Myc expression in the TSLP-stimulated trophoblast proliferation. These results suggest that TSLP may upregulate c-Myc expression through activation of STAT3 pathway, thereby inducing trophoblast proliferation.

Excitation by GABA in the SCN reaches its time and place (Commentary on Irwin & Allen)

The mammalian circadian clock resides within the suprachiasmatic nucleus (SCN). This relatively small group of neurons controls most aspects of our daily lives by dictating our ‘internal time’ and keeping us synchronized with the environment. Intensive research in the past 20 years has revealed that a complex intracellular molecular mechanism enables each of the SCN neurons to function as a stand-alone circadian oscillator (Hastings et al., 2008). The firing rate of these neurons alternates between high and low levels that correspond to the light and dark phases of the circadian rhythm. However, for efficient function of the circadian clock, these individual oscillators must be synchronized. Since the SCN is comprised of interconnected GABAergic neurons, it is tempting to assume that this GABAergic network serves as a synchronizing device. Over a decade ago we reported (Wagner et al., 1997) that in the SCN GABA can operate either as an excitatory or inhibitory neurotransmitter. We further demonstrated the prevalence of excitation in response to GABA during the light phase and predominance of inhibition during the dark phase. We suggested that the duality of GABAergic effect on SCN neurons, which reflects circadian oscillations in intracellular chloride concentrations, serves to amplify the circadian rhythmicity in firing rate. This original report was immediately followed by a joint publication by three groups reporting that GABA has only an inhibitory effect in the SCN (Gribkoff et al., 1999). Since then, however, a steady stream of publications confirmed the dual effect of GABA (De Jeu & Pennartz, 2002; Colwell, 2003; Ikeda et al., 2003; Choi et al., 2008). The groundbreaking study of Albus et al. (2005) has ascribed the duality of GABA effect with a physiological role by demonstrating that the SCN is composed of two well-defined compartments, termed ‘core’ and ‘shell’. It is the GABA-mediated interaction between these two compartments that enable the SCN to respond to light resetting signals. They also found that excitation by GABA is more common in the shell compartment whereas inhibition is more frequently found within the core neurons, probably due to differential expression of the chloride transporter KCC1 (Belenky et al., 2008). The study by Irwin & Allen (2009) published in this issue of EJN adds an important contribution to the duality of the GABA effect in the SCN. They loaded SCN neurons with a calcium indicator that enables simultaneous recordings of calcium concentrations in a large portion of the SCN neuronal network, thus allowing for an accurate description of the distribution of the GABAergic effect in time and space. Their results clearly show that most SCN neurons (50–70%) respond to GABA application with an increase in intracellular calcium concentrations, which is associated with neuronal excitation. A careful analysis of the contribution of GABAA and GABAB receptors confirms that the excitatory effect is mediated solely by the GABAA receptor, which is a chloride channel. Using specific blockers Irwin & Allen (2009) further demonstrate that the change in calcium concentration is caused by voltage-sensitive calcium channels, responding to the GABA-mediated shift in membrane potential. Analysis of the spatial and temporal distribution of the responses to GABA shows that that the excitatory responses prevail, and that they are most abundant in the shell region. Moreover, in both shell and core compartments excitation predominates during the night phase. In order to demonstrate that the duality of the GABA effect occurs within physiological conditions, one must show that it can be induced by endogenous GABA. Irwin & Allen (2009) confirm this by blocking GABAA receptors and by stimulating the optical nerve input to the SCN. Whereas stimulating the optical nerve induces changes in calcium concentration that are similar to those induced by GABA, blocking GABAA receptors induces the opposite effect. Overall these results demonstrate that GABA excitation is far from an anecdotal observation, and that it in fact dominates the network activity of the SCN. Beyond alternating between excitation and inhibition, GABA also imposes a dual effect on the intracellular calcium concentrations. Given the prominent role played by intracellular calcium in numerous cellular processes, this finding opens new avenues for research and interpretations. The most exciting possibility is that the GABA-mediated changes in calcium concentration directly affect gene expression, thus providing the elusive link between neuronal activity and the molecular clock of SCN neurons.

A Crucial Role for p57Kip2 in the Intracellular Timer that Controls Oligodendrocyte Differentiation

The intracellular molecular mechanism that controls the timing of oligodendrocyte differentiation remains unknown. Temple and Raff (1986) previously showed that an oligodendrocyte precursor cell (OPC) can divide a maximum of approximately eight times before its daughter cells simultaneously cease proliferating and differentiate into oligodendrocytes. They postulated that over time the level of an intracellular molecule might synchronously change in each daughter cell, ultimately reaching a level that prohibited additional proliferation. Here, we report the discovery of such a molecule, the cyclin-dependent kinase inhibitor p57(Kip2) (Cdkn1c). We show in vitro that all daughters of a clone of OPCs express similar levels of p57(Kip2), that p57(Kip2) levels increase over time in proliferating OPCs, and that p57(Kip2) levels regulate how many times an OPC can divide before differentiating. These findings reveal a novel part of the mechanism by which OPCs measure time and are likely to extend to similar timers in many other precursor cell types.

Expression of Constitutively Active STAT3 Can Replicate the Cytokine-suppressive Activity of Interleukin-10 in Human Primary Macrophages

There is general agreement that signal transducer and activation of transcription 3 (STAT3) is required to mediate the anti-inflammatory activities of interleukin (IL)-10. However, STAT3 is activated by multiple factors that do not share the anti-inflammatory activity of IL-10. The question remains whether STAT3 is sufficient for the anti-inflammatory effects or whether there are other signals required, as had been suggested previously. We set out to map the human IL-10 receptor and to identify the key elements involved in transducing the cytokine-suppressive effects of IL-10. We were able to show an absolute requirement for both of the tyrosine residues found within the YXXQ-STAT3-docking site within the IL-10 receptor 1 and that no other signals appeared to be required. We used a constitutively active STAT3 to determine whether expression of this factor could suppress lipopolysaccharide-induced tumor necrosis factor and IL-6 production. Our data show that STAT3 activity can suppress both IL-6 and tumor necrosis factor production in lipopolysaccharide-stimulated macrophages. However, in synovial fibroblasts, STAT3 did not suppress IL-6 production, suggesting that the cellular environment plays an important role in dictating whether STAT3 drives a pro- or anti-inflammatory response.

Insulin‐like growth factor‐1 prevents Amyloid beta[25–35]/ (H2O2)‐ induced apoptosis in lymphocytes by reciprocal NF‐kappa‐B activation and p53 inhibition via PI3K‐dependent pathway

The role of insulin-like growth factor (IGF-1) as neural survival factor for the treatment of Alzheimer's disease has recently gained attention. However, the detailed intracellular mechanism involved in this effect remains to be fully elucidated. The present study shows that IGF-1 protects peripheral blood lymphocytes (PBL) from (10, 30 μM) Aβ[25–35] and (25, 50, 100 μM) H2O2-induced apoptosis through NF-κB activation and p53 down regulation via (PI) 3-kinase (PI-3K)–dependent pathway as demonstrated by using either specific (25 μM) LY294002 (PI-3K inhibitor), (10nM) ammonium pyrrolidinedithiocarbamate (PDTC; NF-κB inhibitor), 50nM pifithrin-α (PFT; p53 inhibitor) or by using immunocytochemistry detection of NF-κB and p53 transcription factors activation. Importantly, IGF-1, PDTC and PFT were able to protect and rescue lymphocytes pre-exposed to 10μM Aβ[25–35], even when the three compounds were added up-to 12 h post- Aβ[25–35] exposure. These results suggest that survival/rescue of lymphocytes from Aβ[25–35] toxicity is determined by p53 inactivation via IGF-1/ PI-3K pathway. These data may contribute to a better understanding of the intracellular molecular mechanism by which IGF-1 promotes cell survival against oxidative stress stimuli.

Phosphoinositide Lipid Second Messengers: New Paradigms for Calcium Channel Modulation

Neuronal Ca2+ channels are key transducers coupling excitability to cellular function. As such, they are tightly regulated by multiple G protein-signaling pathways that finely tune their activity. In addition to fast, direct G(beta)gamma modulation of Ca2+ channels, a slower Galpha(q/11)-mediated mechanism has remained enigmatic despite intensive study. Recent work suggests that membrane phosphoinositides are crucial determinants of Ca2+ channel activity. Here, we discuss their role in Ca2+ channel modulation and the leading theories that seek to elucidate the underlying molecular details of the so-called "mysterious" G(q/11)-mediated signal.

Protective effect of vitamin E against focal brain ischemia and neuronal death through induction of target genes of hypoxia-inducible factor-1

Vitamin E has been shown to have protective effects against cerebral ischemia, possibly due to its anti-oxidant effects. However, its non-anti-oxidant, intracellular molecular mechanism remains elusive. For in vivo experiments in rats, orally administered vitamin E significantly reduced not only the brain infarct volume but also space navigation disability after permanent middle cerebral artery (MCA) occlusion. The level of anti-oxidant after MCA occlusion was significantly increased specifically in the ipsilateral brain tissues of vitamin E-treated rats. For in vitro experiments, posttreatment with vitamin E protected primary cultured neurons from nitric oxide-induced insult. Vitamin E induced the expression of the α subunit of hypoxia-inducible factor-1 (HIF-1) and its target genes, including vascular endothelial growth factor (VEGF) and heme oxygenase-1. The hypoxia response element on the VEGF promoter was responsible for this vitamin E-induced transcriptional activation of VEGF gene. Taken together, these results suggest that cerebral infarction increased the permeability of vitamin E across the blood–brain barrier, and this increased vitamin E in brain tissue elicited neuroprotective effects not only through scavenging oxidants, as are previously well reported, but also by transactivating HIF-1-dependent genes, which results in protection of brains from ischemic insults.

Induction of Somatic Mutation in a Human B Cell Line In Vitro

Both the B cell–surface trigger(s) and the intracellular molecular mechanism(s) of somatic hypermutation in immunoglobulin (Ig) variable region genes remain unknown, partly because of the lack of a simple and reproducible in vitro model. Here, we show that upon surface immunoglobulin cross-linking followed by coculture with activated cloned T cells, the Burkitt's lymphoma cell line BL2 is induced to mutate its IgV H gene. Repeated activation of BL2 cells increased the frequency of mutation. The in vitro–induced mutations, which do not affect the IgM constant region, are point mutations distributed over the entire V HDJ H gene segment and do not show evidence of antigen-driven selection.