The condensing vacuole of exocrine cells is more acidic than the mature secretory vesicle

A number of intracellular, membrane-bound compartments in both the endocytic and exocytic pathways of eukaryotic cells have an acidic internal pH. In endocrine cells, the mature secretory vesicle has an acidic pH; secretory vesicles isolated from exocrine cells, however, appear to have a neutral pH. Recently we have used a newly developed immunocytochemical technique to map low-pH compartments in insulin-secreting islet cells with the electron microscope and find that during the maturation of the secretory vesicle there is a progressive acidification of these vesicles that begins as soon as the trans Golgi condensing vacuoles form. Now we have used this technique to examine two exocrine cells: the pancreatic acinar cell and the parotid serous cell. In both cell types, the trans Golgi condensing vacuoles are acidic and accumulate the low-pH probe to the same extent as condensing vacuoles of insulin-secreting islet cells. Unlike insulin-secreting cells, however, maturation of the granules is accompanied by a return of luminal pH to near neutrality. Therefore, although the pH of storage granules in exocrine and endocrine cells is different, the pH of the condensing vacuoles in both cells is acidic.     

Listeriolysin O allows Listeria monocytogenes replication in macrophage vacuoles

Listeria monocytogenes is an intracellular bacterial pathogen that replicates rapidly in the cytosol of host cells during acute infection. Surprisingly, these bacteria were found to occupy vacuoles in liver granuloma macrophages during persistent infection of severe combined immunodeficient (SCID) mice. Here we show that L. monocytogenes can replicate in vacuoles within macrophages. In livers of SCID mice infected for 21 days, we observed bacteria in large LAMP1(+) compartments that we termed spacious Listeria-containing phagosomes (SLAPs). SLAPs were also observed in vitro, and were found to be non-acidic and non-degradative compartments that are generated in an autophagy-dependent manner. The replication rate of bacteria in SLAPs was found to be reduced compared to the rate of those in the cytosol. Listeriolysin O (LLO, encoded by hly), a pore-forming toxin essential for L. monocytogenes virulence, was necessary and sufficient for SLAP formation. A L. monocytogenes mutant with low LLO expression was impaired for phagosome escape but replicated slowly in SLAPs over a 72 h period. Therefore, our studies reveal a role for LLO in promoting L. monocytogenes replication in vacuoles and suggest a mechanism by which this pathogen can establish persistent infection in host macrophages.     

A new mechanism for the neutralization of enveloped viruses by antiviral antibody

Despite the considerable research that has been carried out into viral neutralization by antiviral antibody, its mechanisms remain poorly understood. Cases have been reported in which antiviral antibody can inhibit viral replication without inhibiting the binding and uptake of virus by susceptible cells. It has been shown that many enveloped viruses enter their target cells by endocytosis and are subsequently located in cellular compartments of increasing acidity. With several enveloped viruses this acidic pH can catalyse a fusion reaction between the membrane of the virus particle and that of a prelysosomal endosome, thus enabling the viral core to enter the cytosol and replication to commence. We have recently demonstrated that such an endosomal fusion event at mild acidic pH is involved in the entry pathway of the enveloped flavivirus, West Nile virus (WNV), into macrophages. We now show that antiviral antibody can neutralize WNV by inhibiting this intraendosomal acid-catalysed fusion step and we speculate on possible implications for the future design of antiviral vaccines.     

Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis

Phagocytosis and autophagy are two ancient, highly conserved processes involved, respectively, in the removal of extracellular organisms and the destruction of organisms in the cytosol. Autophagy, for either metabolic regulation or defence, involves the formation of a double membrane called the autophagosome, which then fuses with lysosomes to degrade the contents, a process that has similarities with phagosome maturation. Toll-like-receptor (TLR) engagement activates a variety of defence mechanisms within phagocytes, including facilitation of phagosome maturation, and also engages autophagy. Therefore we speculated that TLR signalling might link these processes to enhance the function of conventional phagosomes. Here we show that a particle that engages TLRs on a murine macrophage while it is phagocytosed triggers the autophagosome marker LC3 to be rapidly recruited to the phagosome in a manner that depends on the autophagy pathway proteins ATG5 and ATG7; this process is preceded by recruitment of beclin 1 and phosphoinositide-3-OH kinase activity. Translocation of beclin 1 and LC3 to the phagosome was not associated with observable double-membrane structures characteristic of conventional autophagosomes, but was associated with phagosome fusion with lysosomes, leading to rapid acidification and enhanced killing of the ingested organism.     

GILT is a critical host factor for Listeria monocytogenes infection

Listeria monocytogenes is a gram-positive, intracellular, food-borne pathogen that can cause severe illness in humans and animals. On infection, it is actively phagocytosed by macrophages; it then escapes from the phagosome, replicates in the cytosol, and subsequently spreads from cell to cell by a non-lytic mechanism driven by actin polymerization. Penetration of the phagosomal membrane is initiated by the secreted haemolysin listeriolysin O (LLO), which is essential for vacuolar escape in vitro and for virulence in animal models of infection. Reduction is required to activate the lytic activity of LLO in vitro, and we show here that reduction by the enzyme gamma-interferon-inducible lysosomal thiol reductase (GILT, also called Ifi30) is responsible for the activation of LLO in vivo. GILT is a soluble thiol reductase expressed constitutively within the lysosomes of antigen-presenting cells, and it accumulates in macrophage phagosomes as they mature into phagolysosomes. The enzyme is delivered by a mannose-6-phosphate receptor-dependent mechanism to the endocytic pathway, where amino- and carboxy-terminal pro-peptides are cleaved to generate a 30-kDa mature enzyme. The active site of GILT contains two cysteine residues in a CXXC motif that catalyses the reduction of disulphide bonds. Mice lacking GILT are deficient in generating major histocompatibility complex class-II-restricted CD4(+) T-cell responses to protein antigens that contain disulphide bonds. Here we show that these mice are resistant to L. monocytogenes infection. Replication of the organism in GILT-negative macrophages, or macrophages expressing an enzymatically inactive GILT mutant, is impaired because of delayed escape from the phagosome. GILT activates LLO within the phagosome by the thiol reductase mechanism shared by members of the thioredoxin family. In addition, purified GILT activates recombinant LLO, facilitating membrane permeabilization and red blood cell lysis. The data show that GILT is a critical host factor that facilitates L. monocytogenes infection.     

酸性环境对斑节对虾的危害及酸化控制

报道演海虾场和文科虾场池水返酸过程，池养斑节对虾对酸性环境反应，池水慢性返酸使斑节对虾发生软壳，维生素Ｃ缺乏症，蜕壳不完全等症状，急性返酸到ｐＨ值６．１１以下时引起对虾突然蜕壳而大量死亡通过对比实验，认为施用石灰提高ｐＨ值，施肥繁殖浮游藻类，利用藻类光合作用消耗二氧化碳，降低酸性，大潮时加大换水量，小潮时抽水提高水位，能有效地防止池水返酸，控制斑节对虾酸性环境综合症。     

Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1

With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome-lysosome fusion through the AS160 (also known as TBC1D4)-RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.     

A candidate NAD+ transporter in an intracellular bacterial symbiont related to Chlamydiae

Bacteria living within eukaryotic cells can be essential for the survival or reproduction of the host but in other cases are among the most successful pathogens. Environmental Chlamydiae, including strain UWE25, thrive as obligate intracellular symbionts within protozoa; are recently discovered relatives of major bacterial pathogens of humans; and also infect human cells. Genome analysis of UWE25 predicted that this symbiont is unable to synthesize the universal electron carrier nicotinamide adenine dinucleotide (NAD+). Compensation of limited biosynthetic capacity in intracellular bacteria is usually achieved by import of primary metabolites. Here, we report the identification of a candidate transporter protein from UWE25 that is highly specific for import of NAD+ when synthesized heterologously in Escherichia coli. The discovery of this candidate NAD+/ADP exchanger demonstrates that intact NAD+ molecules can be transported through cytoplasmic membranes. This protein acts together with a newly discovered nucleotide transporter and an ATP/ADP translocase, and allows UWE25 to exploit its host cell by means of a sophisticated metabolic parasitism.     

The third component of complement (C3) is responsible for the intracellular survival of Leishmania major

Leishmania are obligate intracellular parasites of mononuclear phagocytes. We and others have shown that the promastigote form of all species of leishmania activates complement from non-immune serum and that this activation can result in parasite lysis. This work, as well as earlier in vivo studies, suggested that complement is an important component of host defence against leishmaniasis. We now present evidence that parasite complement fixation, in addition to increasing parasite phagocytosis, is required for the intracellular survival of leishmania in macrophages. We specifically show a strong correlation between parasite C3 fixation and intracellular survival. We attribute this survival, in part, to a decrease in the magnitude of the macrophage respiratory burst which is triggered by complement-coated, as opposed to uncoated, parasites.     

氧化亚铁硫杆菌与硫酸改善城市污泥脱水性能的研究

研究生物酸化和化学酸化调理法对污泥脱水性能的影响.结果表明,生物酸化调理污泥的脱水效果和稳定性均优于化学酸化.在接种氧化亚铁硫杆菌菌液为10%时,可使原泥脱水效率提高49.7%;而酸处理的最佳条件为初始pH 3.2,此时污泥脱水性能较原泥提高30.8%.比较调理过程中污泥体系pH的变化发现,当接种量一定,生物调理法可较长时间维持利于脱水的酸性环境.经电镜扫描可见,生物法调理的污泥含硫杆菌,其团形均匀,孔隙度大,泥质松软.红外光谱分析表明,氧化亚铁硫杆菌通过酶作用把胞外聚合物中的蛋白质分解,从而改善污泥脱水性能.     

重庆地区 20 年间紫色土酸化研究

以酸沉降危害严重的重庆市为研究区域,对其主要农业土壤--紫色土进行大面积土壤酸化调查,与20世纪80年代中期重庆市土壤普查的保留样品进行对比分析研究,以探讨近20年来酸沉降对紫色土的危害程度.结果表明:与20世纪80年代中期比较,目前重庆市农用地和蔬菜地中的石灰性紫色土减少,而酸性和强酸性紫色土增加;同时,农用地的土壤交换性酸和交换性铝均有一定程度的上升.证实了在近20年的酸沉降影响下,重庆市大部分紫色土已发生酸化,且酸化程度日益加深.     

福建省土壤酸化现状与治理对策——以福建省18个县（市、区）为例

以福建省18个县（市、区）为调查位点，对土壤酸化现状及治理情况开展调查。通过实地调查、取样分析和数据收集，获得各调查位点土壤pH值数据。结果表明，18个县（市、区）中，强酸性（pH≤50）土壤占13个，酸性（5.0＜pH≤5.5）土壤占5个。2017—2021年，在福建省的9个设区市中，有7个市的土壤酸化问题依然严重。通过“线上问卷+电话访谈+实地调查”的方式，对地方政府、农户和企业3类调查对象开展调研，摸清了福建省土壤酸化治理现状及面临的问题。研究表明:应高度重视土壤酸化治理工作，充分利用福建省丰富的贝类加工副产物贝壳，将其加工成土壤调理剂，达到治理土壤酸化，缓解土壤重金属污染，提高作物产量和品质，助推经济绿色发展的效果。基于“重协同、强产业、全链条、促发展”的治理理念，结合成功案例，提出政府主导、校企助推、人才助农、精准施策的“政产学研用”多元互助模式，共同推动土壤治理。     

Endothelial tubes assemble from intracellular vacuoles in vivo

The formation of epithelial tubes is crucial for the proper development of many different tissues and organs, and occurs by means of a variety of different mechanisms. Morphogenesis of seamless, properly patterned endothelial tubes is essential for the development of a functional vertebrate circulatory system, but the mechanism of vascular lumenization in vivo remains unclear. Evidence dating back more than 100 years has hinted at an important function for endothelial vacuoles in lumen formation. More than 25 years ago, in some of the first endothelial cell culture experiments in vitro, Folkman and Haudenschild described "longitudinal vacuoles" that "appeared to be extruded and connected from one cell to the next", observations confirmed and extended by later studies in vitro showing that intracellular vacuoles arise from integrin-dependent and cdc42/Rac1-dependent pinocytic events downstream of integrin-extracellular-matrix signalling interactions. Despite compelling data supporting a model for the assembly of endothelial tubes in vitro through the formation and fusion of vacuoles, conclusive evidence in vivo has been lacking, primarily because of difficulties associated with imaging the dynamics of subcellular endothelial vacuoles deep within living animals. Here we use high-resolution time-lapse two-photon imaging of transgenic zebrafish to examine how endothelial tubes assemble in vivo, comparing our results with time-lapse imaging of human endothelial-cell tube formation in three-dimensional collagen matrices in vitro. Our results provide strong support for a model in which the formation and intracellular and intercellular fusion of endothelial vacuoles drives vascular lumen formation.     

Carbon-dioxide-induced exocytotic insertion of H+ pumps in turtle-bladder luminal membrane: role of cell pH and calcium

The contents of endocytic vesicles and other intracellular organelles (such as Golgi and microsomes) are acidified by an electrogenic proton-translocating ATPase that is remarkably similar to that found in urinary epithelia. We recently found that the number of H+ ATPases in the apical plasma membrane of these epithelia is regulated by exocytotic insertion of endocytic vesicles whose membranes contain this H+ pump. Carbon dioxide, a major stimulus for urinary acidification, causes rapid fusion of these vesicles with the luminal membrane, thereby inserting these pumps there and increasing the rate of net transepithelial H+ secretion; CO2 also inhibits endocytic retrieval of the pumps from the luminal membrane. Such reciprocal regulation of endocytosis and exocytosis by a physiological modulator makes this system particularly attractive for studying the cellular events regulating membrane fusion. Here we present evidence that CO2 induces exocytosis by a cascade of events, the first step of which is cytoplasmic acidification. Cell acidification then increases calcium activity, which causes the fusion event.     

伤寒沙门菌非编码RNA AsrC表达特性及功能初步研究

非编码RNA在细菌的各种生命活动中都发挥着重要的功能.针对伤寒沙门菌非编码RNA AsrC,对其转录和降解特性及其所具有的功能进行初步研究.首先使用Northern blot和qRT-PCR的方法检测了σ因子rpoE和rpoS对AsrC转录的影响,分析了使用利福平抑制细菌RNA合成的情况下,RNase E和RNase III对AsrC降解的影响.其次使用AsrC缺陷株和高表达菌株分析细菌在酸、氧、高渗应激条件下的生长情况.再者通过全基因组芯片技术,研究AsrC高表达菌株基因表达的变化.进一步研究AsrC在细菌侵袭和巨噬细胞胞内生存力中的作用.结果显示,rpoE在酸应激下、rpoS在高渗应激下调节AsrC的转录.细菌中RNase E主要参与了AsrC的降解.高表达AsrC后,有40个基因表达上调,23个基因表达下调.AsrC高表达可以增强伤寒沙门菌对于上皮细胞的侵袭力,并且可以减弱细菌的胞内生存和增殖力.     

Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion

The generation of an acidic pH in intracellular organelles is required for several membrane and protein recycling processes. For instance, the internalization of ligands by receptor-mediated endocytosis is followed by the development of an acidic pH inside endosomes; this allows dissociation of the ligand, which is then transported to the lysosomes, from the receptor, which is recycled to the cell surface. There is evidence that part of this recycling process involves the distal region of the Golgi complex, where terminal glycosylation occurs: when the plasma membrane transferrin receptor is desialylated by neuraminidase treatment, it acquires new sialic acid molecules after endocytosis and before cell-surface re-expression. Golgi membranes have been shown to contain a proton pump and the distal Golgi cisternae appear to have an acidic content. Here, we have studied the effects of chloroquine and ammonium chloride, which raise the pH of acidic intracellular compartments, on the processing and secretion of immunoglobulins by plasma cells. Sialic acid transfer to terminal galactose residues, a reaction known to occur in the distal Golgi shortly before secretion, is completely and rapidly inhibited in the presence of these drugs, without significant modification of the secretion rate. This effect is accompanied by a dilatation of the Golgi cisternae and is not rapidly reversible.     

胞外ATP对VK2/E6E7细胞胞内pH的影响

 利用激光扫描共聚焦显微镜（Confocal microscope）采用pH依赖的荧光探针Snarf-4F测量细胞胞内pH(pHi)方法,发现胞外ATP能剂量依赖（10~1 000 μmol/L）地降低人阴道上皮细胞株VK2/E6E7细胞pHi。当胞外加入200 μmol/L ATP时,能快速地使VK2/E6E7细胞pHi降低,此降低的pHi在洗脱ATP后可迅速恢复。这些结果表明胞外ATP能引起VK2/E6E7细胞胞内酸化。     

Volcanic carbon dioxide vents show ecosystem effects of ocean acidification

The atmospheric partial pressure of carbon dioxide (p(CO(2))) will almost certainly be double that of pre-industrial levels by 2100 and will be considerably higher than at any time during the past few million years. The oceans are a principal sink for anthropogenic CO(2) where it is estimated to have caused a 30% increase in the concentration of H(+) in ocean surface waters since the early 1900s and may lead to a drop in seawater pH of up to 0.5 units by 2100 (refs 2, 3). Our understanding of how increased ocean acidity may affect marine ecosystems is at present very limited as almost all studies have been in vitro, short-term, rapid perturbation experiments on isolated elements of the ecosystem. Here we show the effects of acidification on benthic ecosystems at shallow coastal sites where volcanic CO(2) vents lower the pH of the water column. Along gradients of normal pH (8.1-8.2) to lowered pH (mean 7.8-7.9, minimum 7.4-7.5), typical rocky shore communities with abundant calcareous organisms shifted to communities lacking scleractinian corals with significant reductions in sea urchin and coralline algal abundance. To our knowledge, this is the first ecosystem-scale validation of predictions that these important groups of organisms are susceptible to elevated amounts of p(CO(2)). Sea-grass production was highest in an area at mean pH 7.6 (1,827 (mu)atm p(CO(2))) where coralline algal biomass was significantly reduced and gastropod shells were dissolving due to periods of carbonate sub-saturation. The species populating the vent sites comprise a suite of organisms that are resilient to naturally high concentrations of p(CO(2)) and indicate that ocean acidification may benefit highly invasive non-native algal species. Our results provide the first in situ insights into how shallow water marine communities might change when susceptible organisms are removed owing to ocean acidification.