Effects of Hypomagnetic Field on Magnetosome Formation of Magnetospirillum Magneticum AMB-1

Magnetotactic bacteria synthesize intracellular magnetic particles, magnetosomes, which arrange in chain(s) and confer on cell a magnetic dipolar moment. To explore the function of geomagnetic field to magnetotactic bacteria, the effects of hypomagnetic field on magnetosome formation in Magnetospirillum magneticum AMB-1 were studied. Cells were cultivated in a specially designed device where geomagnetic field was reduced by about 100-fold to less than 500nT. AMB-1 cultures were incubated in hypomagnetic field or geomagnetic field. Results showed that hypomagnetic field had no significant effects on the average number of magnetic particles per bacterium and bacterial iron depletion. However, the growth (OD) of cell at stationary-phase was lower and cellular magnetism (R _mag) at exponential growth phase was higher than that of bacteria cultivated in geomagnetic field. Statistic results on transmission electron microscopy (TEM) micrographs showed that the average size of magnetic particles in AMB-1 cells in hypomagnetic field group was larger than that of in geomagnetic field group and more ratio of larger-size magnetic particles (>50 nm) was observed when cultivated 16 h under hypomagnetic field. Furthermore, the influences of hypomagnetic field on gene expression were studied in AMB-1 cells. Quantitative RT-PCR results showed that hypomagnetic field up-regulated mms13, down-regulated mms6 and had no effect on magA. Together, the results showed that hypomagnetic field could affect the growth of AMB-1 at the stationary-phase, the crystallization process of magnetosomes, and mms13, mms6 expressions. In addition, our results suggested that the geomagnetic field plays an important role in the biomineralization of magnetosomes.     

Distinct intrinsic and synaptic properties of pre‐sympathetic and pre‐parasympathetic output neurons in Barrington's nucleus

Barrington's nucleus (BN), commonly known as the pontine micturition center, controls micturition and other visceral functions through projections to the spinal cord. In this study, we developed a rat brain slice preparation to determine the intrinsic and synaptic mechanisms regulating pre‐sympathetic output (PSO) and pre‐parasympathetic output (PPO) neurons in the BN using patch‐clamp recordings. The PSO and PPO neurons were retrogradely labeled by injecting fluorescent tracers into the intermediolateral region of the spinal cord at T13‐L1 and S1‐S2 levels, respectively. There were significantly more PPO than PSO neurons within the BN. The basal activity and membrane potential were significantly lower in PPO than in PSO neurons, and A‐type K⁺ currents were significantly larger in PPO than in PSO neurons. Blocking A‐type K⁺ channels increased the excitability more in PPO than in PSO neurons. Stimulting μ‐opioid receptors inhibited firing in both PPO and PSO neurons. The glutamatergic EPSC frequency was much lower, whereas the glycinergic IPSC frequency was much higher, in PPO than in PSO neurons. Although blocking GABA_A receptors increased the excitability of both PSO and PPO neurons, blocking glycine receptors increased the firing activity of PPO neurons only. Furthermore, blocking ionotropic glutamate receptors decreased the excitability of PSO neurons but paradoxically increased the firing activity of PPO neurons by reducing glycinergic input. Our findings indicate that the membrane and synaptic properties of PSO and PPO neurons in the BN are distinctly different. This information improves our understanding of the neural circuitry and central mechanisms regulating the bladder and other visceral organs.     

Diethylmaleate and iodoacetate in combination caused profound cell death in astrocytes

Energy failure and oxidative stress have been implicated in the pathogenesis of ischemia. Here, we report a potential link between cytosolic phospholipase A₂ (cPLA₂) activation and energy failure/oxidative stress‐induced astrocyte damage involving reactive oxygen species (ROS), protein kinase C‐α (PKC‐α), Src, Raf, and extracellular signal‐regulated kinase (ERK) signaling and concurrent elevation of endogenous chelatable zinc. Energy failure and oxidative stress were produced by treating astrocytes with glycolytic inhibitor iodoacetate and glutathione chelator diethylmaleate, respectively. Diethylmaleate and iodoacetate in combination caused augmented damage to astrocytes in a time‐ and concentration‐dependent manner. The cell death caused by diethylmaleate/iodoacetate was accompanied by increased ROS generation, PKC‐α membrane translocation, Src, Raf, ERK, and cPLA₂ phosphorylation. Pharmacological studies revealed that these activations all contributed to diethylmaleate/iodoacetate‐induced astrocyte death. Intriguingly, the mobilization of endogenous chelatable zinc was observed in diethylmaleate/iodoacetate‐treated astrocytes. Zinc appears to act as a downstream mediator in response to diethylmaleate/iodoacetate treatment because of the attenuating effects of its chelator N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine. These observations indicate that ROS/PKC‐α, Src/Raf/ERK signaling and cPLA₂ are active participants in diethylmaleate/iodoacetate‐induced astrocyte death and contribute to a vicious cycle between the depletion of ATP/glutathione and the mobilization of chelatable zinc as critical upstream effectors in initiating cytotoxic cascades.

     

Functional Investigation of Transmembrane Helix 3 in H+-Translocating Pyrophosphatase

H⁺-translocating pyrophosphatase (H⁺-PPase, EC 3.6.1.1) plays an important role in acidifying vacuoles by transporting protons across membranes at the expense of pyrophosphate (PP_i) hydrolysis. Vigna radiata H⁺-PPase (VrH⁺-PPase) contains 16 transmembrane helices (TMs). The hydrophobicity of TM3 is relatively lower than that of most other TMs, and the amino acids in this TM are highly conserved in plants. Furthermore, TM5 and -6, which are the core TMs involving in H⁺-PPase functions, are near TM3. It is thus proposed that TM3 is associated with H⁺-PPase activity. To address this possibility, site-directed mutagenesis was applied in this investigation to determine the role of TM3 in VrH⁺-PPase. Upon alanine/serine substitution, T138 and S142, whose side chains face toward the center TMs, were found to be involved in efficient proton transport. G149/S153 and G160/A164 pairs at the crucial termini of the two GxxxG-like motifs are indispensable in maintaining enzymatic activities and conformational stability. Moreover, stability in the vicinity surrounding G149 is pivotal for efficient expression. S153, M161 and A164 are critical for the K⁺-mediated stimulation of H⁺-PPase. Taken together, our results demonstrate that TM3 plays essential roles in PP_i hydrolysis, proton transport, expression, and K⁺ stimulation of H⁺-PPase.     

Extracellular Signal-regulated Kinase 5 (ERK5) Mediates Prolactin-stimulated Adult Neurogenesis in the Subventricular Zone and Olfactory Bulb

Prolactin-stimulated adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) mediates several reproductive behaviors including mating/pregnancy, dominant male pheromone preference in females, and paternal recognition of offspring. However, downstream signaling mechanisms underlying prolactin-induced adult neurogenesis are completely unknown. We report here for the first time that prolactin activates extracellular signal-regulated kinase 5 (ERK5), a MAP kinase that is specifically expressed in the neurogenic regions of the adult mouse brain. Knockdown of ERK5 by retroviral infection of shRNA attenuates prolactin-stimulated neurogenesis in SVZ-derived adult neural stem/progenitor cells (aNPCs). Inducible erk5 deletion in adult neural stem cells of transgenic mice inhibits neurogenesis in the SVZ and OB following prolactin infusion or mating/pregnancy. These results identify ERK5 as a novel and critical signaling mechanism underlying prolactin-induced adult neurogenesis.     

O-GlcNAcylation of Cofilin Promotes Breast Cancer Cell Invasion

O-GlcNAcylation is a post-translational modification that regulates a broad range of nuclear and cytoplasmic proteins and is emerging as a key regulator of various biological processes. Previous studies have shown that increased levels of global O-GlcNAcylation and O-GlcNAc transferase (OGT) are linked to the incidence of metastasis in breast cancer patients, but the molecular basis behind this is not fully known. In this study, we have determined that the actin-binding protein cofilin is O-GlcNAcylated by OGT and mainly, if not completely, mediates OGT modulation of cell mobility. O-GlcNAcylation at Ser-108 of cofilin is required for its proper localization in invadopodia at the leading edge of breast cancer cells during three-dimensional cell invasion. Loss of O-GlcNAcylation of cofilin leads to destabilization of invadopodia and impairs cell invasion, although the actin-severing activity or lamellipodial localization is not affected. Our study provides insights into the mechanism of post-translational modification in fine-tuning the regulation of cofilin activity and suggests its important implications in cancer metastasis.     

Ubiquitin E3 Ligase CRL4CDT2/DCAF2 as a Potential Chemotherapeutic Target for Ovarian Surface Epithelial Cancer

Cullin-RING ubiquitin ligases (CRLs) are the largest family of E3 ligases and require cullin neddylation for their activation. The NEDD8-activating enzyme inhibitor MLN4924 reportedly blocked cullin neddylation and inactivated CRLs, which resulted in apoptosis induction and tumor suppression. However, CRL roles in ovarian cancer cell survival and the ovarian tumor repressing effects of MLN4924 are unknown. We show here that CRL4 components are highly expressed in human epithelial ovarian cancer tissues. MLN4924-induced DNA damage, cell cycle arrest, and apoptosis in ovarian cancer cells in a time- and dose-dependent manner. In addition, MLN4924 sensitized ovarian cancer cells to other chemotherapeutic drug treatments. Depletion of CRL4 components Roc1/2, Cul4a, and DDB1 had inhibitory effects on ovarian cancer cells similar to MLN4924 treatment, which suggested that CRL4 inhibition contributed to the chemotherapeutic effect of MLN4924 in ovarian cancers. We also investigated for key CRL4 substrate adaptors required for ovarian cancer cells. Depleting Vprbp/Dcaf1 did not significantly affect ovarian cancer cell growth, even though it was expressed by ovarian cancer tissues. However, depleting Cdt2/Dcaf2 mimicked the pharmacological effects of MLN4924 and caused the accumulation of its substrate, CDT1, both in vitro and in vivo. MLN4924-induced DNA damage and apoptosis were partially rescued by Cdt1 depletion, suggesting that CRL4^CDT2 repression and CDT1 accumulation were key biochemical events contributing to the genotoxic effects of MLN4924 in ovarian cancer cells. Taken together, these results indicate that CRL4^CDT2 is a potential drug target in ovarian cancers and that MLN4924 may be an effective anticancer agent for targeted ovarian cancer therapy.     

EXTL2, a Member of the EXT Family of Tumor Suppressors,Controls Glycosaminoglycan Biosynthesis in a Xylose Kinase-dependent Manner

Mutant alleles of EXT1 or EXT2, two members of the EXT gene family, are causative agents in hereditary multiple exostoses, and their gene products function together as a polymerase in the biosynthesis of heparan sulfate. EXTL2, one of three EXT-like genes in the human genome that are homologous to EXT1 and EXT2, encodes a transferase that adds not only GlcNAc but also N-acetylgalactosamine to the glycosaminoglycan (GAG)-protein linkage region via an α1,4-linkage. However, both the role of EXTL2 in the biosynthesis of GAGs and the biological significance of EXTL2 remain unclear. Here we show that EXTL2 transfers a GlcNAc residue to the tetrasaccharide linkage region that is phosphorylated by a xylose kinase 1 (FAM20B) and thereby terminates chain elongation. We isolated an oligosaccharide from the mouse liver, which was not detected in EXTL2 knock-out mice. Based on structural analysis by a combination of glycosidase digestion and 500-MHz ¹H NMR spectroscopy, the oligosaccharide was found to be GlcNAcα1-4GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate), which was considered to be a biosynthetic intermediate of an immature GAG chain. Indeed, EXTL2 specifically transferred a GlcNAc residue to a phosphorylated linkage tetrasaccharide, GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate). Remarkably, the phosphorylated linkage pentasaccharide generated by EXTL2 was not used as an acceptor for heparan sulfate or chondroitin sulfate polymerases. Moreover, production of GAGs was significantly higher in EXTL2 knock-out mice than in wild-type mice. These results indicate that EXTL2 functions to suppress GAG biosynthesis that is enhanced by a xylose kinase and that the EXTL2-dependent mechanism that regulates GAG biosynthesis might be a “quality control system” for proteoglycans.     

LY2228820 Dimesylate,a Selective Inhibitor of p38 Mitogen-activated Protein Kinase,Reduces Angiogenic Endothelial Cord Formation in Vitro and in Vivo

LY2228820 dimesylate is a highly selective small molecule inhibitor of p38α and p38β mitogen-activated protein kinases (MAPKs) that is currently under clinical investigation for human malignancies. p38 MAPK is implicated in a wide range of biological processes, in particular those that support tumorigenesis. One such process, angiogenesis, is required for tumor growth and metastasis, and many new cancer therapies are therefore directed against the tumor vasculature. Using an in vitro co-culture endothelial cord formation assay, a surrogate of angiogenesis, we investigated the role of p38 MAPK in growth factor- and tumor-driven angiogenesis using LY2228820 dimesylate treatment and by shRNA gene knockdown. p38 MAPK was activated in endothelial cells upon growth factor stimulation, with inhibition by LY2228820 dimesylate treatment causing a significant decrease in VEGF-, bFGF-, EGF-, and IL-6-induced endothelial cord formation and an even more dramatic decrease in tumor-driven cord formation. In addition to involvement in downstream cytokine signaling, p38 MAPK was important for VEGF, bFGF, EGF, IL-6, and other proangiogenic cytokine secretion in stromal and tumor cells. LY2228820 dimesylate results were substantiated using p38α MAPK-specific shRNA and shRNA against the downstream p38 MAPK effectors MAPKAPK-2 and HSP27. Using in vivo models of functional neoangiogenesis, LY2228820 dimesylate treatment reduced hemoglobin content in a plug assay and decreased VEGF-A-stimulated vascularization in a mouse ear model. Thus, p38α MAPK is implicated in tumor angiogenesis through direct tumoral effects and through reduction of proangiogenic cytokine secretion via the microenvironment.