Extracellular calcium does not contribute to cryopreservation-induced cytotoxicity

Summary The possible role of extracellular calcium ([Ca⁺²]e) in cryopreservation-induced cytotoxicity was tested using Madin-Darby canine kidney (MDCK) cells and a fluorescent multiple endpoint assay. MDCK cells maintained in 2 mM [Ca⁺²]e and treated with the calcium ionophore, ionomycin, increased their intracellular calcium ([Ca⁺²]i) as revealed by the calcium indicator dye, Fluo3 and the bottom-reading spectrofluorometer, CytoFluor 2300. The addition of 10 mM [ethylene bis (oxyethylenenitrilo)]-tetraacetic acid (EGTA) to the extracellular medium before treatment with ionomycin blocked this ionomycin-dependent increase in [Ca⁺²]i. A number of site and activity-specific fluorescent probes were surveyed to determine which indicator dye might best reveal the ionomycin-induced cytotoxic events during this increase in [Ca⁺²]i. Although most dyes changed their emission profiles in response to calcium, neutral red was found to best reflect the loss of [Ca⁺²]i homeostasis. The NR50 for a 15-min exposure to ionomycin in the presence of 2 mM [Ca⁺²]e was approximately 2μM ionomycin, but ionomycin had little apparent effect on neutral red retention when 10 mM EGTA was added to the extracellular medium. Thus it was clear that an increase in [Ca⁺²]i could be cytotoxic to MDCK cells and that neutral red could monitor this cytotoxic episode. To test if [Ca⁺²]e was similarly cytotoxic during cryopreservation, MDCK cells were subjected to cryopreservation in the presence of dimethylsulfoxide (DMSO). In contrast to previous studies, plasma membrane integrity, not lysosomal function, seemed to best correlate with cell survival subsequent to cryopreservation. In addition, decreasing [Ca⁺²]e had no discernable effect on the retention of plasma membrane indicator dyes, neutral red, or cell survival. It is concluded that a) plasma membrane indicator dyes, not neutral red, might be better indicators of cytotoxicity occurring during cryopreservation; b) DMSO might be toxic to lysosomes during cryopreservation of cultured cells; and c) although [Ca⁺²]e can contribute to cytotoxicity, the presence of [Ca⁺²]e might not influence cryopreservation-induced cytotoxicity.     

NADPH oxidases contribute to autophagy regulation

Reactive oxygen species (ROS) are emerging as regulators of autophagy in various cellular contexts. There are many cellular sources of ROS in eukaryotic cells. In phagocytes, the critical immune cells for host defense, the Nox2 NADPH oxidase generates ROS during phagocytosis and plays a central role in microbial killing. Toll-like receptors (TLRs) are important membrane microbial sensing receptors, which can activate Nox2,¹ and were recently demonstrated to signal autophagy targeting of phagosomes to promote their maturation.² Our recent study reveals that Nox2 activity and its generated ROS are key signals that induce TLR-activated autophagy of phagosomes. Our results provide the first evidence that ROS from the Nox2 NADPH oxidase can contribute to regulating autophagy in host defense against bacteria. The association of TLR, Nox2 and autophagy with inflammatory bowel disease (IBD) suggests a significant role of this antibacterial pathway in these diseases.     

Multiple cellular responses to serotonin contribute to epithelial homeostasis

Epithelial homeostasis incorporates the paradoxical concept of internal change (epithelial turnover) enabling the maintenance of anatomical status quo. Epithelial cell differentiation and cell loss (cell shedding and apoptosis) form important components of epithelial turnover. Although the mechanisms of cell loss are being uncovered the crucial triggers that modulate epithelial turnover through regulation of cell loss remain undetermined. Serotonin is emerging as a common autocrine-paracine regulator in epithelia of multiple organs, including the breast. Here we address whether serotonin affects epithelial turnover. Specifically, serotonin's roles in regulating cell shedding, apoptosis and barrier function of the epithelium. Using in vivo studies in mouse and a robust model of differentiated human mammary duct epithelium (MCF10A), we show that serotonin induces mammary epithelial cell shedding and disrupts tight junctions in a reversible manner. However, upon sustained exposure, serotonin induces apoptosis in the replenishing cell population, causing irreversible changes to the epithelial membrane. The staggered nature of these events induced by serotonin slowly shifts the balance in the epithelium from reversible to irreversible. These finding have very important implications towards our ability to control epithelial regeneration and thus address pathologies of aberrant epithelial turnover, which range from degenerative disorders (e.g.; pancreatitis and thyrioditis) to proliferative disorders (e.g.; mastitis, ductal ectasia, cholangiopathies and epithelial cancers).     

Two photic pathways contribute to pineal evoked responses

In this study, average photic evoked responses were recorded simultaneously in freely behaving rats from the pineal body and the ventromedial hypothalamus; permanent semimicroelectrodes were implanted several days before the experiments were begun, and both local anesthesia (xylocaine), sympathectomy, and general anesthesia (barbiturate) were used as tools to find out whether or not photic responses are transmitted to the pineal via the superior cervical ganglion (scg)-nervi conorii and/or through another CNS route. The experiments demonstrated that the photic evoked responses recorded from the pineal are transmitted via two separate routes: one, a fast pathway with a “shorter” latency, via the CNS, i.e., the habencular posterior commissure complex, and the other a “slower” (or longer) pathway via the scg to the pineal.     

Extracellular matrix regulation of autophagy

Integrin-mediated attachment of epithelial cells to extracellular matrix (ECM) is crucial for proper growth and survival. Although detachment leads to apoptosis, termed anoikis, recent work demonstrates that ECM detachment also robustly induces autophagy, a tightly regulated lysosomal self-digestion process that actually promotes survival. Autophagy presumably protects epithelial cells from the stresses of ECM detachment, allowing them to survive provided that they reattach in a timely manner. Currently, the intracellular signals linking integrin engagement to autophagy remain unclear, but certain growth factor, energy-sensing, and stress-response pathways represent attractive candidates. Moreover, autophagy may be a previously unrecognized mechanism utilized by detached cancer cells to survive anoikis, which may facilitate tumor cell dormancy, dissemination, and metastasis.     

Interactions within the intrinsic cardiac nervous system contribute to chronotropic regulation

The objective of this study was to determine how neurons within the right atrial ganglionated plexus (RAGP) and posterior atrial ganglionated plexus (PAGP) interact to modulate right atrial chronotropic, dromotropic, and inotropic function, particularly with respect to their extracardiac vagal and sympathetic efferent neuronal inputs. Surgical ablation of the PAGP (PAGPx) attenuated vagally mediated bradycardia by 26%; it reduced heart rate slowing evoked by vagal stimulation superimposed on sympathetically mediated tachycardia by 36%. RAGP ablation (RAGPx) eliminated vagally mediated bradycardia, while retaining the vagally induced suppression of sympathetic-mediated tachycardia (-83%). After combined RAGPx and PAGPx, vagal stimulation still reduced sympathetic-mediated tachycardia (-47%). After RAGPx alone and after PAGPx alone, stimulation of the vagi still produced negative dromotropic effects, although these changes were attenuated compared with the intact state. Negative dromotropic responses to vagal stimulation were further attenuated after combined ablation, but parasympathetic inhibition of atrioventricular nodal conduction was still demonstrable in most animals. Finally, neither RAGPx nor PAGPx altered autonomic regulation of right atrial inotropic function. These data indicate that multiple aggregates of neurons within the intrinsic cardiac nervous system are involved in sinoatrial nodal regulation. Whereas parasympathetic efferent neurons regulating the right atrium, including the sinoatrial node, are primarily located within the RAGP, prejunctional parasympathetic-sympathetic interactions regulating right atrial function also involve neurons within the PAGP.     

Vascular cells contribute to atherosclerosis by cytokine- and innate-immunity-related inflammatory mechanisms

Cardiovascular diseases are the human diseases with the highest death rate and atherosclerosis is one of the major underlying causes of cardiovascular diseases. Inflammatory and innate immune mechanisms, employing monocytes, innate receptors, innate cytokines, or chemokines are suggested to be involved in atherogenesis. Among the inflammatory pathways the cytokines are central players. Plasma levels of cytokines and related proteins, such as CRP, have been investigated in cardiovascular patients, tissue mRNA expression was analyzed and correlations to vascular diseases established. Consistent with these findings the generation of cytokine-deficient animals has provided direct evidence for a role of cytokines in atherosclerosis. In vitro cell culture experiments further support the suggestion that cytokines and other innate mechanisms contribute to atherogenesis. Among the initiation pathways of atherogenesis are innate mechanisms, such as toll-like-receptors (TLRs), including the endotoxin receptor TLR4. On the other hand, innate cytokines, such as IL-1 or TNF, or even autoimmune triggers may activate the cells. Cytokines potently activate multiple functions relevant to maintain or spoil homeostasis within the vessel wall. Vascular cells, not least smooth muscle cells, can actively contribute to the inflammatory cytokine-dependent network in the blood vessel wall by: (i) production of cytokines; (ii) response to these potent cell activators; and (iii) cytokine-mediated interaction with invading cells, such as monocytes, T-cells, or mast cells. Activation of these pathways results in accumulation of cells and increased LDL- and ECM-deposition which may serve as an 'immunovascular memory' resulting in an ever-growing response to subsequent invasions. Thus, vascular cells may potently contribute to the inflammatory pathways involved in development and acceleration of atherosclerosis.     

白三烯B4受体对类风湿关节炎的免疫调节作用

目的：白三烯B4（LTB4）受体包括BLT1和BLT2两个亚型,利用各自的特异性拮抗剂U-75302、LY255283,通过体外实验分析LTB4受体在炎症免疫反应中介导的作用。方法：①以U-75302和LY255283分别阻断BLT1和BLT2,用OH—TdR掺入法检测大鼠滑膜细胞的增殖情况。②流式细胞术检测U-75302或LY255283对大鼠脾CD4^＋T细胞中IFN-γ和IL-4表迭的影响,分析Th1、Th2细胞的百分率。结果：①U-75302仅在浓度为10μmol／L时方可检测到对滑膜细胞增殖的明显抑制作用（P〈0．05）,而LY255283在10nmol／L～10μmol／L范围内对滑膜细胞均有明显的抑制作用（P〈0．05）。②流式细胞术分析结果表明,当U-75302或LY255283达到10μmol／L时,可明显升高Th2细胞的百分率,而对Th1细胞的百分率影响不明显。结论：BLT1和（或）BLT2介入了大鼠滑膜细胞增殖、Th1／Th2淋巴细胞百分率变化,对于类风湿关节炎（rheumatoid arthritis,RA）等炎症疾病的防治而言,BLT1、BLT2可能具有重要的药物靶点意义。     

NK cells contribute to intracellular bacterial infection-mediated inhibition of allergic responses

To experimentally examine the hygiene hypothesis, here we studied the effect of chlamydial infection on the development of allergic responses induced by OVA and the involvement of NK cells in this process using a mouse model of airway inflammation. We found that prior Chlamydia muridarum infection can inhibit airway eosinophilic inflammation and mucus production induced by allergen sensitization and challenge. The inhibition was correlated with an alteration of allergen-driven cytokine-producing patterns of T cells. We demonstrated that NK cells were activated following chlamydial infection, showing both cell expansion and cytokine secretion. The in vivo depletion of NK cells using anti-NK Ab before OVA sensitization and challenge partially abolished the inhibitory effect of chlamydial infection, which was associated with a partial restoration of Th2 cytokine production. In contrast, the adoptive transfer of NK cells that were isolated from infected mice showed a significant inhibitory effect on allergic responses, similar to that observed in natural infection. The data suggest that the innate immune cells such as NK cells may play an important role in infection-mediated inhibition of allergic responses.     

Extracellular calcium participates in responses to acetylcholine in Xenopus oocytes

We tested the contribution of extracellular calcium (Ca2+) to membrane electrical responses to acetylcholine (ACh) in native Xenopus oocytes. Removal of Cao caused a decrease in both the rapid (D1) and the slow (D2) chloride currents that comprise the common depolarizing response to ACh in native oocyte. The effect of Ca2+o removal on the muscarinic response was mimicked by the addition of 1 mM Mn2+, an effective antagonist of calcium influx, though not by antagonists of voltage-sensitive calcium channels. When oocytes were challenged with ACh in Ca2(+)-free medium, subsequent addition of 1.8 mM CaCl2 resulted in a rapid, often transient, depolarizing current. Similarly to the Ca2+o-dependent component of membrane electrical responses, the Ca2(+)-evoked current was reversibly abolished by Mn2+, though not by antigonists of voltage-sensitive calcium channels. Depletion of cellular calcium potentiated the Ca2(+)-evoked current, implying negative feedback of calcium channels by calcium. Injection of 10-100 fmol of inositol 1,4,5-trisphosphate (IP3) resulted in a two-component depolarizing current. IP3 injection promoted the appearance of Ca2+o-evoked current that was significantly potentiated by previous calcium depletion. We suggest that activation of cell-membrane muscarinic receptors causes opening of apparently voltage-insensitive and verapamil or diltiazem-resistant calcium channels. These channels may be activated by IP3 or its metabolites, which increase following the activation of cell membrane receptors coupled to a phospholipase C. The channels may be identical to receptor-operated channels described in other model systems.     

成年小鼠前脑NMDA受体参与神经元的动作电位发放

谷氨酸是中枢神经系统主要的快速兴奋性递质。AMPA受体和海人藻酸受体主要参与突触传递,而NMDA受体主要参与突触可塑性。基因操作的方法增强NMDA受体的功能,可以增强动物在正常生理状态下的学习能力,及在组织损伤情况下的反应敏感性。NMDA受体参与生理功能的主要机制是长时程增强（long—term potentiation,LTP）。我们的研究表明,NMDA受体不仅参与刺激前扣带皮层的第五层细胞或刺激白质诱导的突触反应,而且参与在胞体施加去极化跃阶电流诱导的动作电位的发放。钙一钙调蛋白敏感的腺苷酸环化酶1（adenylyl cyclase 1,AC1）和cAMP信号通路可能介导了这些反应。由于扣带皮层神经元在伤害性刺激和痛中发挥重要作用,我们的结果为前脑NMDA受体参与突触传递和动作电位发放,以及与前脑相关的行为,如感受伤害性刺激和痛,提供了一个新的机制。     

Endosomal protein sorting and autophagy genes contribute to the regulation of yeast life span

Accumulating evidence from various organisms points to a role for autophagy in the regulation of life span. By performing a genome-wide screen to identify novel life span determinants in Saccharomyces cerevisiae, we have obtained further insights into the autophagy-related and -unrelated degradation processes that may be important for preventing cellular senescence. The generation of multivesicular bodies and their fusion with the vacuole in the endosomal pathway emerged as novel cell functions involved in yeast chronological survival and longevity extension.     

Endogenous retroviruses promote homeostatic and inflammatory responses to the microbiota

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Neuropeptide regulation of inflammatory and immunologic responses. The capacity of alpha-melanocyte-stimulating hormone to inhibit tumor necrosis factor and IL-1-inducible biologic responses

Administration of the pituitary hormone alpha-melanocyte-stimulating hormone (alpha-MSH) to mice was found to inhibit a number of IL-1 and TNF-inducible biologic responses in situ. The ability of either IL-1 or TNF to cause fever, enhance plasma levels of acute phase proteins, and increase the numbers of peripheral blood neutrophils was inhibited by the simultaneous peripheral administration of this neuropeptide. In addition, alpha-MSH reversed the depressive influences of IL-1 or TNF on the effector phase of contact hypersensitivity (CH) responses in animals given an adoptive transfer of primed lymphocytes from hapten-sensitized donors. Intracerebral injection of nanogram quantities of alpha-MSH inhibited the ability of peripherally administered IL-1 or TNF to induce both fever and neutrophilia without affecting the increase in plasma levels of serum amyloid P and fibrinogen. Also, nanogram quantities of alpha-MSH given intracerebrally to normal mice did not reverse the depressed CH responses observed after peripheral IL-1 or TNF administration. These findings suggest that both fever and neutrophilia are linked to the direct action of IL-1 or TNF on the brain. This was supported by the observation that an intracerebral injection of IL-1 or TNF in low doses increased core body temperature and circulating neutrophil numbers without affecting plasma levels of acute phase proteins or CH responsiveness. Our results provide additional support for the hypothesis that bidirectional control exists between elements of the neuroendocrine and immune systems.