Role of Intercellular and Intracellular Communication by Nitric Oxide in Coupling of Muscarinic Receptors to Activation of Guanylate Cyclase in Neuronal Cells

Abstract: Muscarinic receptor-mediated cyclic GMP formation and release of nitric oxide (NO) (or a precursor thereof) were compared in mouse neuroblastoma N1E-115 cells. [³H]Cyclic GMP was assayed in cells prelabeled with [³H]guanine. Release of NO upon the addition of muscarinic agonists to unlabeled neuroblastoma cells (NO donor cells) was quantitated indirectly by its ability to increase the [³H]cyclic GMP level in labeled cells whose muscarinic receptors were inactivated by irreversible alkylation (NO detector cells). Carbachol increased NO release in a concentration-dependent manner, with half-maximal stimulation at 173 μ M (compared to 96 μ M for direct activation of cyclic GMP formation). The maximal effect of carbachol in stimulating release of NO when measured indirectly was lower than that in elevating [³H]cyclic GMP directly in donor cells. Hemoglobin was more effective in blocking the actions of released NO than in attenuating direct stimulation of [³H]cyclic GMP synthesis. There was a good correlation between the ability of a series of muscarinic agonists to release NO or to activate [³H]cyclic GMP formation directly, and the potency of pirenzepine in inhibiting the two responses. Furthermore, there was a similar magnitude of desensitization of both responses by prolonged receptor activation or stimulation of protein kinase C. NO release was also regulated in relation to the cellular growth phase. A model is proposed in which a fraction of NO generated upon receptor activation does not diffuse extracellularly and stimulates cyclic GMP synthesis within the same cell where it is formed (locally acting NO). The remainder of NO that is extruded extracellularly might travel to neighboring cells (neurotransmitter NO) or might be taken back into the cells of origin (homing NO).     

古菌RecJ蛋白的研究进展

RecJ蛋白属于aspartate-histidine-histidine (DHH)磷酸酯酶超家族,存在于细菌、真核生物和古菌中。细菌RecJ蛋白是一种5′→3′ssDNA外切酶,参与错配修复、同源重组、碱基切除修复等生物学过程。真核生物cell division cycle 45 (Cdc45)蛋白是细菌RecJ核酸酶的同源物,但不具有核酸酶活性。Cdc45蛋白能够与minichromosomemaintenance(MCM)和Go-Ichi-Ni-San(GINS)形成Cdc45-MCM-GINS (CMG)复合物,是真核生物DNA复制的重要组分。在古菌中,几乎所有基因组已测序的古菌均编码一种或多种RecJ蛋白同源物。与细菌RecJ核酸酶不同,古菌RecJ蛋白具有多样化的核酸酶活性,并且能够与MCM和GINS形成类似于真核生物CMG的复合物。因此,古菌RecJ蛋白是参与古菌DNA复制、修复和重组的重要成分。基于目前古菌RecJ蛋白的研究报道,本文综述了古菌RecJ蛋白的活性、结构与功能方面的研究进展,聚焦于不同古菌RecJ蛋白以及它们与细菌RecJ核酸酶和真核生物RecJ同源物的...     

αCT1 peptide sensitizes glioma cells to temozolomide in a glioblastoma organoid platform

Glioblastoma (GBM) is the most common form of brain cancer. Even with aggressive treatment, tumor recurrence is almost universal and patient prognosis is poor because many GBM cell subpopulations, especially the mesenchymal and glioma stem cell populations, are resistant to temozolomide (TMZ), the most commonly used chemotherapeutic in GBM. For this reason, there is an urgent need for the development of new therapies that can more effectively treat GBM. Several recent studies have indicated that high expression of connexin 43 (Cx43) in GBM is associated with poor patient outcomes. It has been hypothesized that inhibition of the Cx43 hemichannels could prevent TMZ efflux and sensitize otherwise resistance cells to the treatment. In this study, we use a three-dimensional organoid model of GBM to demonstrate that combinatorial treatment with TMZ and αCT1, a Cx43 mimetic peptide, significantly improves treatment efficacy in certain populations of GBM. Confocal imaging was used to visualize changes in Cx43 expression in response to combinatorial treatment. These results indicate that Cx43 inhibition should be pursued further as an improved treatment for GBM.     

Regulation of neuronal nitric oxide synthase by histone,protamine, and myelin basic protein

We examined the effects of endogenous basic proteins rich in the amino acidL-arginine on neuronal NO synthase activity by monitoring cyclic GMP formation in intact neuron-like neuroblastoma N1E-115 cells. Histone, protamine and myelin basic protein significantly stimulated cyclic GMP formation, both in a time- and concentration-dependent manner. These effects were blocked by hemoglobin and NO synthase inhibitors. Removal of the extracellular/intracellular Ca²⁺ gradient by a Ca²⁺ chelator completely abolished the cyclic GMP responses elicited by histone and protamine, suggesting that influex of extracellular Ca²⁺ might be involved in their activation of NO synthase. The effects of myelin basic protein on cyclic GMP formation, however, appeared to be due to Ca²⁺ release from intracellular stores. In cytosolic preparations of rat cerebellum, these basic proteins inhibited the metabolism ofL-arginine intoL-citrulline by NO synthase. We conclude from our findings that endogenous basic proteins might be involved in the regulation of neuronal NO synthase activity. Their effects on the enzyme could be either stimulatory or inhibitory, depending on whether the basic proteins exert their effects extracellularly or intracellularly, respectively.     

Coordinated regulation of anthranilate metabolism and bacterial virulence by the GntR family regulator MpaR in Pseudomonas aeruginosa

The GntR family regulators are widely distributed in bacteria and play critical roles in metabolic processes and bacterial pathogenicity. In this study, we describe a GntR family protein encoded by PA4132 that we named MpaR (M vfR-mediated P QS and a nthranilate r egulator) for its regulation of Pseudomonas quinolone signal (PQS) production and anthranilate metabolism in Pseudomonas aeruginosa. The deletion of mpaR increased biofilm formation and reduced pyocyanin production. RNA sequencing analysis revealed that the mRNA levels of antABC encoding enzymes for the synthesis of catechol from anthranilate, a precursor of the PQS, were most affected by mpaR deletion. Data showed that MpaR directly activates the expression of mvfR, a master regulator of pqs system, and subsequently promotes PQS production. Accordingly, deletion of mpaR activates the expression of antABC genes, and thus, increases catechol production. We also demonstrated that MpaR represses the rhl quorum-sensing (QS) system, which has been shown to control antABC activity. These results suggested that MpaR function is integrated into the QS regulatory network. Moreover, mutation of mpaR promotes bacterial survival in a mouse model of acute pneumonia infection. Collectively, this study identified a novel regulator of pqs system, which coordinately controls anthranilate metabolism and bacterial virulence in P. aeruginosa.     

药用白及资源再生策略初探

白及为兰科白及属植物。作为著名中药,白及功效多样、用途广泛,具有较高的药用价值和经济价值。随资源利用强度的增加,野生白及资源日渐萎缩,人工规模化种植是实现白及药用资源可持续利用的必由之路。优良种质及繁育技术是种植业的关键问题。为了更好地推动白及资源研究和可持续利用,本文就白及种质及快繁技术研究进展作一综述,并就白及资源再生策略进行初步探讨,为相关研究和生产提供参考。     

MDP25 mediates the fine-tuning of microtubule organization in response to salt stress

Microtubules are dynamic cytoskeleton structures playing fundamental roles in plant responses to salt stress. The precise mechanisms by which microtubule organization is regulated under salt stress are largely unknown. Here, we report that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN 25 (MDP25; also known as PLASMA MEMBRANE-ASSOCIATED CATION-BINDING PROTEIN 1 (PCaP1)) helps regulate microtubule organization. Under salt treatment, elevated cytosolic Ca²⁺ concentration caused MDP25 to partially dissociate from the plasma membrane, promoting microtubule depolymerization. When Ca²⁺ signaling was blocked by BAPTA-AM or LaCl₃, microtubule depolymerization in wild-type and MDP25-overexpressing cells was slower, while there was no obvious change in mdp25 cells. Knockout of MDP25 improved microtubule reassembly and was conducive to microtubule integrity under long-term salt treatment and microtubule recovery after salt stress. Moreover, mdp25 seedlings exhibited a higher survival rate under salt stress. The presence microtubule-disrupting reagent oryzalin or microtubule-stabilizing reagent paclitaxel differentially affected the survival rates of different genotypes under salt stress. MDP25 promoted microtubule instability by affecting the catastrophe and rescue frequencies, shrinkage rate and time in pause phase at the microtubule plus-end and the depolymerization rate at the microtubule minus-end. These findings reveal a role for MDP25 in regulating microtubule organization under salt treatment by affecting microtubule dynamics.     

Role of caveolin-1 in epidermal stem cells during burn wound healing in rats

Local transplantation of stem cells has therapeutic effects on skin damage but cannot provide satisfactory wound healing. Studies on the mechanisms underlying the therapeutic effects of stem cells on skin wound healing will be needed. Hence, in the present study, we explored the role of Caveolin-1 in epidermal stem cells (EpiSCs) in the modulation of wound healing. We first isolated EpiSCs from mouse skin tissues and established stable EpiSCs with overexpression of Caveolin-1 using a lentiviral construct. We then evaluated the epidermal growth factor (EGF)-induced cell proliferation ability using cell counting Kit-8 (CCK-8) assay and assessed EpiSC pluripotency by examining Nanog mRNA levels in EpiSCs. Furthermore, we treated mice with skin burn injury using EpiSCs with overexpression of Caveolin-1. Histological examinations were conducted to evaluate re-epithelialization, wound scores, cell proliferation and capillary density in wounds. We found that overexpression of Caveolin-1 in EpiSCs promoted EGF-induced cell proliferation ability and increased wound closure in a mouse model of skin burn injury. Histological evaluation demonstrated that overexpression of Caveolin-1 in EpiSCs promoted re-epithelialization in wounds, enhanced cellularity, and increased vasculature, as well as increased wound scores. Taken together, our results suggested that Caveolin-1 expression in the EpiSCs play a critical role in the regulation of EpiSC proliferation ability and alteration of EpiSC proliferation ability may be an effective approach in promoting EpiSC-based therapy in skin wound healing.     

Chitosan combined with sodium silicate treatment induces resistance against rot caused by Alternaria alternata in postharvest jujube fruit

The effect of chitosan (0.1 mol/L) combined with sodium silicate (100 mmol/L) treatment on Alternaria rot caused by Alternaria alternata in postharvest jujube fruit (Ziziphus jujuba Mill. cv. Dongzao) was studied. The results showed that chitosan combined with sodium silicate treatment significantly reduced the lesion diameter, decay incidence, red index and weight loss of jujube fruit compared with control samples. Combining treatment increased the ascorbic acid, flavonoids, total phenolic compounds and lignin content. The level of superoxide anion () and hydrogen peroxide of treated samples was also increased compared with the control samples. Meanwhile, the activities of phenylalanine ammonia lyase, polyphenol oxidase, superoxide dismutase, peroxidase, chitinase and β‐1,3‐glucanase were also accumulated in treated jujube samples, while the activity of catalase markedly decreased. These results indicated that chitosan combined with sodium silicate treatment could induce the disease resistance of postharvest jujube. Therefore, coating postharvest jujube using chitosan combined with sodium silicate could promise as a novel method for preventing the disease infection of postharvest jujube.