非生物胁迫对烟草悬浮细胞胞内和胞外ATP水平的影响

以烟草悬浮细胞BY-2(Nicotiana tabacum ‘Bright Yellow-2’)为材料,研究了NaCl、PEG(6000)、低温3种非生物胁迫对细胞内ATP(intracellular ATP,iATP)和细胞外ATP(extracellular ATP,eATP)水平的影响。结果显示：50～200 mmol·L^-1 NaCl处理导致烟草悬浮细胞膜通透性显著增加(P<0.05),100和200 mmol·L^-1 NaCl处理时iATP和eATP水平显著降低(P<0.05)。随着PEG质量浓度的增加(50、100、200 g·L^-1),烟草悬浮细胞膜通透性和eATP水平逐渐增加,其中在200 g·L^-1 PEG处理时eATP水平显著增加至对照的3.4倍(P<0.05),而iATP水平则在200 g·L^-1 PEG处理时显著降低至对照的0.5倍(P<0.05)。0～10℃低温处理后,烟草悬浮细胞膜通透性和iATP水平呈不同程度增加,其中0℃处...     

Bacterial Versus Archaeal Origin of Extracellular Enzymatic Activity in the Northeast Atlantic Deep Waters

We determined the total and dissolved extracellular enzymatic activity (EEA) of α-glucosidase and β-glucosidase (AGase and BGase), alkaline phosphatase (APase) and leucine aminopeptidase (LAPase) activities in the epi-, meso- and bathypelagic waters of the subtropical Northeast Atlantic. EEA was also determined in treatments in which bacterial EEA was inhibited by erythromycin. Additionally, EEA decay experiments were performed with surface and deep waters to determine EEA lifetimes in both water masses. The proportion of dissolved to total EEA (66–89 %, 44–88 %, 57–82 % and 86–100 % for AGase, BGase, APase and LAPase, respectively) was generally higher than the cell-associated (i.e., particulate) EEA. The percentage of dissolved to total EEA was inversely proportional to the percentage of erythromycin-inhibited to total EEA. Since erythromycin-inhibited plus dissolved EEA equaled total EEA, this tentatively suggests that cell-associated EEA in the open oceanic water column is almost exclusively of bacterial origin. The decay constants of dissolved EEA were in the range of 0.002–0.048 h^?1 depending on the type of extracellular enzyme, temperature and depth in the water column. Although dissolved EEA can have different origins, the major contribution of Bacteria to cell-associated EEA and the long life-time of dissolved EEA suggest that Bacteria—and not mesophilic Archaea—are essentially the main producers of EEA in the open subtropical Northeast Atlantic down to bathypelagic layers.     

MicroRNA-155 Promotes Autophagy to Eliminate Intracellular Mycobacteria by Targeting Rheb

Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3′-untranslated region of Ras homologue enriched in brain (Rheb), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.     

Apparatus for Preparation of Bacterial Extracellular Enzymes

A method is described for the growth of bacteria within dialysis tubing to yield extracellular enzymes, or, possibly, other nondialyzable extracellular products, in concentrated form.     

Fluctuations in the Concentration of Extracellular ATP Synchronized with Intracellular Ca2+ Oscillatory Rhythm in Cultured Cardiac Myocytes

Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically. The intracellular concentration of free Ca²⁺ also changes rhythmically in association with the rhythmic contraction of myocytes (Ca²⁺ oscillation). Both the contraction and Ca²⁺ oscillatory rhythms are synchronized among myocytes, and intercellular communication via gap junctions has been considered primarily responsible for the synchronization. However, a recent study has demonstrated that intercellular communication via extracellular ATP‐purinoceptor signaling is also involved in the intercellular synchronization of intracellular Ca²⁺ oscillation. In this study, we aim to elucidate whether the concentration of extracellular ATP changes cyclically and contributes to the intercellular synchronization of Ca²⁺ oscillation among myocytes. In almost all the cultured cardiac myocytes at four days in vitro (4 DIV), intracellular Ca²⁺ oscillations were synchronized with each other. The simultaneous measurement of the concentration of extracellular ATP and intracellular Ca²⁺ revealed the extracellular concentration of ATP actually oscillated concurrently with the intracellular Ca²⁺ oscillation. In addition, power spectrum and cross‐correlation analyses suggested that the treatment of cultured cardiac myocytes with suramin, a blocker of P2 purinoceptors, resulted in the asynchronization of Ca²⁺ oscillatory rhythms among cardiac myocytes. Treatment with suramin also resulted in a significant decrease in the amplitudes of the cyclic changes in both intracellular Ca²⁺ and extracellular ATP. Taken together, the present study demonstrated the possibility that the concentration of extracellular ATP changes cyclically in association with intracellular Ca²⁺, contributing to the intercellular synchronization of Ca²⁺ oscillation among cultured cardiac myocytes.     

Extracellular Factors of Bacterial Adaptation to Unfavorable Environmental Conditions

Data on extracellular compounds of bacteria involved in their adaptation to unfavorable environmental conditions are reviewed, including high or low temperatures, growth-inhibiting or bactericidal concentrations of toxic substances (oxidants, phenols, and heavy metals) and antibiotics, deviation of pH values from optimum levels, and salinity of the medium. Chemically, the compounds identified belong to diverse types (proteins, hydrocarbons, organic acids, nucleotides, amino acids, lipopeptides, volatile substances, etc.). Most of them remain unidentified, and their properties are studied using biological testing. It has been proposed to view extracellular adaptation factors (EAFs) as a new group of biologically active substances. EAFs may be divided into several subgroups by the mechanism of action. These subgroups include protectors (stabilizers), signaling molecules inducing defense responses, regulators (e.g., adhesion regulators) not acting as inducers, and antidotes (neutralizers). The fields of EAF study include screening (search for new compounds using biological tests), identification, and research into the mechanisms of action. EAFs may find use in biotechnology, medicine, agriculture, and environmental protection.     

Intracellular ATP Concentration Contributes to the Cytotoxic and Cytoprotective Effects of Adenosine

Extracellular adenosine (Ade) interacts with cells by two pathways: by activating cell surface receptors at nanomolar/micromolar concentrations; and by interfering with the homeostasis of the intracellular nucleotide pool at millimolar concentrations. Ade shows both cytotoxic and cytoprotective effects; however, the underlying mechanisms remain unclear. In the present study, the effects of adenosine-mediated ATP on cell viability were investigated. Adenosine treatment was found to be cytoprotective in the low intracellular ATP state, but cytotoxic under the normal ATP state. Adenosine-mediated cytotoxicity and cytoprotection rely on adenosine-derived ATP formation, but not via the adenosine receptor pathway. Ade enhanced proteasome inhibition-induced cell death mediated by ATP generation. These data provide a new pathway by which adenosine exerts dual biological effects on cell viability, suggesting an important role for adenosine as an ATP precursor besides the adenosine receptor pathway.     

Quad-Type Stain for the Simultaneous Demonstration of Intracellular and Extracellular Tissue Components

This technic for the simultaneous demonstration of several different tissue components works equally well on invertebrate and vertebrate tissue if they have been treated with nonchromate fixatives Sections 4-7 μ thick are stained 30 min in 1% Alcian blue, then treated with alkaline alcohol for 2 hr. They are stained in Verhoeff's hematoxylin for 4-6 hr, and rinsed in alcohol; stained in woodstain scarlet-acid fuchsin for 3 min, decolorized in 5% phosphotungstic acid for 20 min and finally stained 5-8 min in alcoholic saffron. Collagen and bone are stained yellow; elastin, myelin and nucleic acids, purple to black; muscle, chitin, cytoplasm, fibrinoid and acid secretion, bright red to lavender; ground substances and mucus, blue-green. Fibrous connective tissue, cartilage, bone and glandular epithelia are exceptionally well demonstrated by this method. Slides stained in this manner are well suited for color photomicrography and as demonstrations in the classroom.     

Extracellular ATP as a signaling molecule for epithelial cells

The charge of this invited review is to present a convincing case for the fact that cells release their ATP for physiological reasons. Many of our "purinergic" colleagues as well as ourselves have experienced resistance to this concept, because it is teleologically counter-intuitive. This review serves to integrate the three main tenets of extracellular ATP signaling: ATP release from cells, ATP receptors on cells, and ATP receptor-driven signaling within cells to affect cell or tissue physiology. First principles will be discussed in the Introduction concerning extracellular ATP signaling. All possible cellular mechanisms of ATP release will then be presented. Use of nucleotide and nucleoside scavengers as well as broad-specificity purinergic receptor antagonists will be presented as a method of detecting endogenous ATP release affecting a biological endpoint. Innovative methods of detecting released ATP by adapting luciferase detection reagents or by using "biosensors" will be presented.Because our laboratory has been primarily interested in epithelial cell physiology and pathophysiology for several years, the role of extracellular ATP in regulation of epithelial cell function will be the focus of this review. For ATP release to be physiologically relevant, receptors for ATP are required at the cell surface. The families of P2Y G protein-coupled receptors and ATP-gated P2X receptor channels will be introduced. Particular attention will be paid to P2X receptor channels that mediate the fast actions of extracellular ATP signaling, much like neurotransmitter-gated channels versus metabotropic heptahelical neurotransmitter receptors that couple to G proteins. Finally, fascinating biological paradigms in which extracellular ATP signaling has been implicated will be highlighted. It is the goal of this review to convert and attract new scientists into the exploding field of extracellular nucleotide signaling and to convince the reader that extracellular ATP is indeed a signaling molecule.     

Studies on Enzymatic Production of ATP

A study of the microflora of radurized Vienna sausages which did not contain preservatives revealed gram-negative diplococci and paired short rods. These radio-resistant isolates were the main residual flora in radurized viennas. The cultures were aerobic, oxidase positive and non-motile, and sugars were oxidized or not attacked at all. The population at the low temperature incubation from 0 to 10°C was abundant and these isolates may be psychrophilic bacteria. The cultures are also capable of growing in media supplemented with 7.5% sodium chloride. The present study has demonstrated that all of these organisms should be tentatively classified as an Intermediate type of Moraxella and Acinetobacter.

D₁₀ values of typical isolates ranged from 44 to 54 krad when irradiated in 0.067 m phosphate buffer, pH 7.0, and were about 4 times larger than that of Acinetobacter calcoaceticus.     

胞外和胞内菌胞外囊泡的功能及应用

细胞外囊泡(Extracellular Vesicles,EVs)是从细胞膜上脱落或者分泌的双层膜结构的囊泡状小体.真核生物、细菌、古细菌和支原体等具有细胞结构的生物均能够释放EVs.细菌分泌的EVs含有DNA、RNA及蛋白质等多种成分,其在细菌毒力保持、免疫逃逸、细菌间物质运输、宿主细胞免疫调节、宿主转录基因调节、耐...     

Bacterial Pathogens Commandeer Rab GTPases to Establish Intracellular Niches

Intracellular bacterial pathogens deploy virulence factors termed effectors to inhibit degradation by host cells and to establish intracellular niches where growth and differentiation take place. Here, we describe mechanisms by which human bacterial pathogens (including Chlamydiae; Coxiella burnetii; Helicobacter pylori; Legionella pneumophila; Listeria monocytogenes; Mycobacteria; Pseudomonas aeruginosa, Salmonella enterica) modulate endocytic and exocytic Rab GTPases in order to thrive in host cells. Host cell Rab GTPases are critical for intracellular transport following pathogen phagocytosis or endocytosis. At the molecular level bacterial effectors hijack Rab protein function to: evade degradation, direct transport to particular intracellular locations and monopolize host vesicles carrying molecules that are needed for a stable niche and/or bacterial growth and differentiation. Bacterial effectors may serve as specific receptors for Rab GTPases or as enzymes that post‐translationally modify Rab proteins or endosomal membrane lipids required for Rab function. Emerging data indicate that bacterial effector expression is temporally and spatially regulated and multiple virulence factors may act concertedly to usurp Rab GTPase function, alter signaling and ensure niche establishment and intracellular bacterial growth, making this field an exciting area for further study.      

Intracellular Trapping of CycloSal-Pronucleotides by Enzymatic Cleavage

A new synthesis for cycloSal-pronucleotides bearing enzymatically cleavable triggers is presented. This trigger is introduced to trap the pronucleotide inside cells. The general concept and hydrolysis data in different media are discussed.     

Extracellular ATP in activity-dependent remodeling of the neuromuscular junction

Electrical activity during early development affects the development and maintenance of synapses (Spitzer [2006]: Nature 4447:707-712), but the intercellular signals regulating maintenance of synapses are not well identified. At the neuromuscular junction, adenosine 5-triphosphate (ATP) is coreleased with acetylcholine at activated nerve terminals to modulate synaptic function. Here we use cocultured mouse motor neurons and muscle cells in a three-compartment cell culture chamber to test whether endogenously released ATP plays a role in activity-dependent maintenance of neuromuscular synapses. The results suggest that ATP release at the synapse counters the negative effect of electrical activity, thus stabilizing activated synapses. Confirming our previous work (Li et al. [2001]: Nat Neurosci 4:871-872), we found that in doubly innervated muscles, electrical stimulation induced heterosynaptic downregulation of the nonstimulated convergent input to the muscle fiber with no or little change of the stimulated inputs. However, in preparations that were stimulated in the presence of apyrase, an enzyme that degrades extracellular ATP, synapse downregulation of stimulated inputs was substantial and significant, and end plate potentials were reduced. Apyrase treatment for 20 h in the absence of stimulation did result in moderate diminution, but this was prevented by blocking spontaneous neural activity with tetrodotoxin. The P2 receptor blocker, suramin, also induced activity-dependent synapse diminution. The decrease in synaptic efficacy produced by prolonged stimulation in the presence of apyrase persisted for greater than 20 h, consistent with a developmental time-course and distinct from the rapid neuromodulatory actions of ATP that have been demonstrated by others. We conclude that extracellular ATP promotes stabilization of the neuromuscular junction and may play a role in activity-dependent synaptic modification during development.     

Comparison of the Lipids of Intracellular and Extracellular Rabies Viruses

The lipid composition of both intracellular and extracellular forms of the ERA strain of rabies virus grown in BHK/21 cells was determined. The lipids from purified preparations of both intracellular and extracellular virus yielded 57 and 58% neutral lipid, respectively. The phospholipids of the intracellular and extracellular virus constituted 43 and 42%, respectively. Triglyceride and cholesterol appear to be the major neutral lipids, whereas sphingomyelin, phosphatidylethanolamine, and phosphatidylcholine comprise the major bulk of phospholipid in both virus types. The molar ratio of cholesterol to phospholipid was 0.87 (intracellular) and 0.92 (extracellular). On the basis of the data presented, it is reasonable to assume that the lipids of both intracellular and extracellular rabies virus are similar.