Mechanism of acid tolerance in a rhizobium strain isolated from Pueraria lobata (Willd.) Ohwi

The Rhizobium sp. strain PR389 was isolated from the root nodules of Pueraria lobata (Willd.) Ohwi, which grows in acidic (pH 4.6) yellow soil of the Jinyun Mountains of Beibei, Chongqing, China. While rhizobia generally have a pH range of 6.5-7.5 for optimum growth, strain PR389 grew in a liquid yeast extract - mannitol agar medium at pH 4.6, as well as in a pH 4.1 soil suspension, suggesting acid tolerance in this specific strain of rhizobium . However, at pH 4.6, the lag phase before vigorous growth was 40 h compared with 4 h under neutral conditions (pH 7.0). For PR389, the generation time after the lag phase remained the same at different pH levels despite the different durations of the lag phase. Except in the pH 4.4 treatment, the pH of the culturing media increased from 4.6, 4.8, 5.0, and 5.5 to neutral and slightly alkaline after 70 h of culture. Chloramphenicol was added to determine if protein production was involved in the increasing pH process. Chloramphenicol significantly inhibited PR389 growth under acid stress but had little effect under neutral conditions. Proton flux measured during a short acid shock (pH 3.8) revealed that this strain has an intrinsic ability to prevent H(+) from entering cells when compared with acid-sensitive rhizobia. We propose that the mechanism for acid tolerance in PR389 involves both intracellular and extracellular processes. When the extracellular pH is lower than pH 4.4, the cell membrane blocks hydrogen from entering the cell. When the pH exceeds 4.4, the rhizobium strain has the ability to raise the extracellular pH, thereby, potentially decreasing the toxicity of aluminum in acid soil.     

Effect of Xyloglucan Oligosaccharides on Growth,Viscoelastic Properties,and Long-Term Extension of Pea Shoots

The growth-promoting effect of xyloglucan-derived oligosaccharides was investigated using a bioassay with entire pea (Pisum sativum L., var Alaska) shoots. After a 24-h incubation period at 25[deg]C, xyloglucan oligosaccharide (XGO) solutions with concentrations of 10-6 M notably increased the growth rate of pea shoots, whereas the same oligosaccharides at 10-7 M were less effective. To investigate the possible correlation between growth rate changes in the XGO-treated shoots and changes in the wall mechanical properties of their growing regions (third internodes), we used a short-term creep assay. The promotion of elongation by XGOs was reflected in an enhancement of the viscoelasticity of the growing regions of the shoots. To show whether this effect on wall viscoelastic properties was the cause or a consequence of their growth promotion, we tested the effect of XGOs on the long-term extension of isolated cell walls. We characterized an acid-induced extension in isolated cell walls from pea shoots that was not inhibited by preincubation in neutral buffers. Exogenously added XGOs did not alter the pattern of pea segment extension at any pH tested, indicating that XGOs have no direct effect on cell wall viscoelasticity. Finally, preincubation of pea segments in neutral buffers with XGOs enhanced their capacity to extend under acidic conditions. This finding suggests that XGOs at a neutral pH can act via transglycosylation, weakening the wall matrix and making the wall more responsive to other mechanisms of acid-induced extension as an expansin-mediated extension.     

Acute effects of extreme pH and its influences on the survival and biochemical biomarkers of juvenile White Shrimp,Litopenaeus vannamei

The White Shrimp (Litopenaeus vannamei) is reared in several types of systems. Changes in pH in some can produce sub-lethal effects that result in poor growth and survival. The aim of the present study was to determine the acidic and basic pH 50% lethal limits after 96 h exposure (pH_50–96 h) and to evaluate the sub-lethal effects of pH on oxidative stress parameters in juvenile L. vannamei. The experimental design comprised nine treatments with three replications of each. The nine treatments consisted of pH 10.5, 10.0, 9.5, 9.0, 7.0, 5.0, 4.5, 4.0 and 3.5. The treatment at 7.0 was used as the control. The sub-lethal pH tests were: basic pH 9.5, neutral pH 7.0 (control) and acidic pH 4.5. For each experimental pH and sampling point, the hemolymph of six shrimp was collected to determine catalase and glutathione S-transferase’s activities and total antioxidant capacity. The values of lethal pH₅₀ at 24, 48, 72 and 96 h for juvenile L. vannamei had basic pH values equal to 9.82, 9.62, 9.59 and 9.58 and acidic pH values of 3.86, 3.92, 3.94 and 4.04, respectively. The shrimp exposed to basic and acidic pH levels showed antioxidant responses with changes in antioxidant activity.     

Vesicular stomatitis virus-mediated cell fusion subsequent to virus adsorption at different pH values.

Vesicular stomatitis virus (VSV)-mediated cell fusion from without can be induced by transient exposure to low pH, subsequent to adsorption of VSV at neutral pH. To study the mechanism of VSV-induced cell fusion, we examined the effect of pH condition at virus adsorption on acid-inducible VSV-mediated cell fusion. Although the binding of VSV to BHK-21 cells was most efficient under acidic condition (pH 5.7-6.3), extensive cell fusion was not observed under this condition. A temporary exposure to low pH after binding at neutral pH also decreased fusion activity. However, return to neutral pH for 2 min just after the acid binding restored the fusion activity. These results indicate the requirement of neutral pH condition for VSV-mediated cell fusion prior to the acid stimulation which induces conformational change of the virus glycoprotein into a fusogenic form.     

Infectious Cell Entry Mechanism of Influenza Virus

Interaction between influenza virus WSN strain and MDCK cells was studied by using spin-labeled phospholipids and electron microscopy. Envelope fusion was negligibly small at neutral pH but greatly activated in acidic media in a narrow pH range around 5.0. The half-time was less than 1 min at 37°C at pH 5.0. Virus binding was almost independent of the pH. Endocytosis occurred with a half-time of about 7 min at 37°C at neutral pH, and about 50% of the initially bound virus was internalized after 1 h. Electron micrographs showed binding of virus particles in coated pits in the microvillous surface of plasma membrane and endocytosis into coated vesicles. Chloroquine inhibited virus replication. The inhibition occurred when the drug was added not later than 10 min after inoculation. Chloroquine caused an increase in the lysosomal pH 4.9 to 6.1. The drug did not affect virus binding, endocytosis, or envelope fusion at pH 5.0. Electron micrographs showed many virus particles remaining trapped inside vacuoles even after 30 min at 37°C in the presence of drug, in contrast to only a few particles after 10 min in vacuoles and secondary lysosomes in its absence. Virus replication in an artificial condition, i.e., brief exposure of the inoculum to acidic medium followed by incubation in neutral pH in the presence of chloroquine, was also observed. These results are discussed to provide a strong support for the infection mechanism of influenza virus proposed previously: virus uptake by endocytosis, fusion of the endocytosed vesicles with lysosome, and fusion of the virus envelope with the surrounding vesicle membrane in the secondary lysosome because of the low pH. This allows the viral genome to enter the target cell cytoplasm.     

Vesicular stomatitis virus binds and fuses with phospholipid domain in target cell membranes

Fusion of vesicular stomatitis virus with some cells (HELR 66, KB, and human erythrocytes, both intact and trypsinized) and liposomes made of various natural and synthetic lipids was studied with spin-labeled phospholipid. Binding of virus was assayed separately with radiolabeled and spin-labeled virus. Binding to cells and liposomes was small at neutral pH but enhanced at acidic pHs. Fusion with cells and liposomes was negligibly small at neutral pH but greatly activated at acidic pHs lower than 6.5. Activation of fusion occurred at lower pH values than enhancement of binding. Fusion occurred rapidly and efficiently, reaching a plateau at 50-80% after 3 min at 37 degrees C. Binding and fusion with cells were enhanced by pretreatment of cells with trypsin. Binding to liposomes was dependent on the head group of the phospholipid, stronger to phosphatidylserine than to phosphatidylcholine, but not much dependent on the acyl chain composition. On the other hand, cis-unsaturated acyl chains were required for the efficient fusion, but there was only a small, if any, requirement for the head group. Cholesterol enhanced the fusion further. High fusion efficiency with cis-unsaturated phospholipids cannot be ascribed to the membrane fluidity but may be related to higher tail-to-head volume ratios. Possible mode of interaction of viral G glycoprotein with phospholipid is discussed. The virus cell entry mechanism is suggested as binding to the phospholipid domain in the cell surface membranes, endocytosis, and followed by fusion with the phospholipid domain in endosomes upon acidification.     

L-pyroglutamate spontaneously formed from L-glutamate inhibits growth of the hyperthermophilic archaeon Sulfolobus solfataricus

Identification of physiological and environmental factors that limit efficient growth of hyperthermophiles is important for practical application of these organisms to the production of useful enzymes or metabolites. During fed-batch cultivation of Sulfolobus solfataricus in medium containing L-glutamate, we observed formation of L-pyroglutamic acid (PGA). PGA formed spontaneously from L-glutamate under culture conditions (78 degrees C and pH 3.0), and the PGA formation rate was much higher at an acidic or alkaline pH than at neutral pH. It was also found that PGA is a potent inhibitor of S. solfataricus growth. The cell growth rate was reduced by one-half by the presence of 5.1 mM PGA, and no growth was observed in the presence of 15.5 mM PGA. On the other hand, the inhibitory effect of PGA on cell growth was alleviated by addition of L-glutamate or L-aspartate to the medium. PGA was also produced from the L-glutamate in yeast extract; the PGA content increased to 8.5% (wt/wt) after 80 h of incubation of a yeast extract solution at 78 degrees C and pH 3.0. In medium supplemented with yeast extract, cell growth was optimal in the presence of 3.0 g of yeast extract per liter, and higher yeast extract concentrations resulted in reduced cell yields. The extents of cell growth inhibition at yeast extract concentrations above the optimal concentration were correlated with the PGA concentration in the culture broth. Although other structural analogues of L-glutamate, such as L-methionine sulfoxide, glutaric acid, succinic acid, and L-glutamic acid gamma-methyl ester, also inhibited the growth of S. solfataricus, the greatest cell growth inhibition was observed with PGA. We also observed that unlike other glutamate analogues, N-acetyl-L-glutamate enhanced the growth of S. solfataricus. This compound was stable under cell culture conditions, and replacement of L-glutamate with N-acetyl-L-glutamate in the medium resulted in increased cell density.     

Colony formations in a halotolerantBrevibacterium sp. JCM 6894 on solid medium with different pH values

Viable cells of a halotolerantBrevibacterium sp. JCM 6894 grown in a liquid medium with pH 7.1 were enumerated as the colony-forming cells on three kinds of agar media with different pH values. Unexpectedly they were lower at neutral pH rather than acidic or alkaline pH. This tendency was invariable regardless of the changes in the concentrations of nutrients in the agar medium as well as in the growth phases of the cells. From the comparison of cell growth between liquid and solid media with different pHs, we notified the importance of the pH changes in liquid medium accompanied with growth. Effects of salts and pH of the liquid medium on protonmotive force (Δp) was estimated from membrane potentials (ΔΨ) and proton gradients (ΔpH) of the strain JCM 6894. In the absence of salts, Δp of the strain JCM 6894 was the largest at neutral pH, which was conflicting with the result of cell viability. The addition of NaCl led to the reduction of Δp at acidic pH, mainly due to the dissipation of ΔΨ, which seems to be consistent with the lower numbers of colony formed at acidic pH in the presence of NaCl.     

Effect of pH on the activity of bovicin HC5, a bacteriocin from Streptococcus bovis HC5

The bacteriocin, bovicin HC5, catalyzed potassium efflux from Streptococcus bovis JB1, and this activity was highly pH dependent. When the pH was near neutral, glucose-energized cells were not affected by bovicin HC5, but the intracellular steady-state concentration of potassium decreased at acidic pH values. The idea that pH was affecting bovicin HC5 binding was supported by the observation that acidic pH also enhanced the efflux of potassium from non-energized cells that had been loaded with potassium. The relationship between bovicin HC5 concentration and potassium depletion was a saturation function, but cooperativity plots indicated that the binding of one bovicin molecule to the cell membrane facilitated the binding of another.     

Visualization of vacuoles in Aspergillus oryzae by expression of CPY-EGFP

Vacuolar carboxypeptidase Y (CPY) from Aspergillus nidulans was used to construct a CPY-EGFP fusion protein and expressed in A. oryzae to study vacuolar morphology and functions in A. oryzae. While the fluorescence of EGFP was barely detectable in A. oryzae expressing CPY-EGFP grown under normal conditions at pH 5-6, the increase in pH of the growth medium towards alkalinity restored the fluorescence. In accordance with such an observation, the fluorescence of CPY-EGFP fusion protein in cell extract decreased in acidic pH condition, concomitant with lowered content of EGFP detected in A. oryzae grown under acidic pH conditions. The pH sensitivity of EGFP fluorescence and enhanced degradation of proteins in vacuoles under acidic pH conditions are thus proposed to result in the reduction of fluorescence in A. oryzae. Further, visualization of vacuoles revealed the presence of peculiar ring- or tube-like structures as distinct from normal spherical-shaped vacuoles.     

Acid-activated insulin-like growth factor binding protein protease activity of Cathepsin D in normal and malignant prostatic epithelial cells and seminal plasma

In this study, we demonstrate insulin-like growth factor binding protein (IGFBP) acid proteolysis in conditioned media (CM) from normal and malignant primary cultures of prostatic epithelial cells, prostatic cell lines, and in seminal plasma. We further demonstrate the absence of such activity in CM from prostatic stromal cells. Radio-labeled IGFBPs (1–6) were incubated with various acidified CM and seminal plasma. None of these media showed IGFBP proteolytic activity at neutral pH, but all CM from prostatic epithelial cells (PC-E) demonstrated strong IGFBP proteolysis at acidic pH. No acid-activated proteolysis was observed in the CM from stromal cell cultures. In order to ascertain the role of cathepsin D, anti-cathepsin antibodies were used to immunodeplete the media of the selected enzymes prior to incubation with IGFBPs. Depletion of cathepsin D greatly reduced the proteolytic activity of the PC-E CM. Additionally, purified cathepsin D yielded a digestion pattern identical to that produced by prostatic cell CM and seminal plasma, following acidic incubation with IGFBP-3. Remarkably, the proteolytic pattern generated by seminal plasma, when incubated with IGFBP-3 at neutral pH, corresponded to that produced by prostate-specific antigen (PSA), demonstrating the interpolation of both neutral and acid proteases from prostate cells into seminal plasma. In conclusion, prostatic epithelial cells secrete acid-specific IGFBP protease(s) related to cathepsin D. Although no significant statistical difference was observed in the degree of acid-specific proteolysis in the media from normal versus malignant primary epithelial cell cultures, physiologicalcharacteristics of the malignant state might facilitate increased cathepsin D activity. We suspect this proteolysis may play a role in prostatic cell proliferationand invasive tumor growth. J. Cell. Physiol. 171:196–204, 1997. © 1997 Wiley-Liss, Inc.     

高温和啶虫脒处理西花蓟马对其F1 代生命表参数的联合作用

【目的】西花蓟马 Frankliniella occidentalis (Pergande) (缨翅目: 蓟马科）是一种危险的入侵害虫,其生长发育受温度影响显著。我们的前期研究表明,高温热激对西花蓟马的杀灭效果并不理想,但高温热激可以改变西花蓟马的药剂敏感性。为了探究高温热激后再进行杀虫剂减量处理能否提高高温对西花蓟马的防治效果,本实验测定了45℃高温热激 2 h后恢复不同时间（8 h 和24 h）啶虫脒对西花蓟马F₁代生命表参数的影响,从控制种群发展的角度探究高温和啶虫脒防治西花蓟马最佳结合方式。【方法】应用特定年龄 龄期及两性生命表的方法,研究45℃高温热激和啶虫脒处理西花蓟马后其F₁代种群的生命表参数。【结果】45℃热激2 h后恢复不同时间用啶虫脒处理西花蓟马亲代,其F₁代卵、1龄幼虫和蛹的平均发育历期均显著长于对照（仅45℃热激2 h）的西花蓟马F₁代（P<0.01）;而且其F₁代雌成虫的寿命和产卵量均显著少于对照（P<0.01）。热激恢复8 h后啶虫脒处理西花蓟马亲代,其F₁代发育历期和雌成虫的寿命虽然与热激恢复24 h的F₁代不存在显著性差异,但是其F₁代的平均产卵前期（adult pre-oviposition period, APOP）和平均总产卵前期（total pre-oviposition period, TPOP）显著长于恢复24 h的F₁代（P<0.01）,单雌平均产卵量显著小于恢复24 h的F₁代（P<0.01）。【结论】相比单一高温防治,高温和杀虫剂综合使用对西花蓟马有更好的防控效果。相比热激后恢复24 h,热激后恢复8 h再进行杀虫剂处理对西花蓟马有更好的防控效果。     

Effect of pH on high-temperature production of bacterial penicillin acylase in Escherichia coli

High-temperature-oriented production of bacterial penicillin acylase (PAC), which is usually expressed at low temperatures (less than 30 degrees C), was demonstrated in this study via heterologous expression of the Providencia rettgeri (P. rettgeri) pac gene in Escherichia coli (E. coli). While it is possible to produce PAC at a temperature as high as 37 degrees C, the environmental condition (specifically, culture pH) critically affected culture performance. Production of PAC at 37 degrees C was feasible only when culture pH was close to neutral (i.e., 6.5-7.5). Outside this pH range, cell physiology for the host/vector system was seriously affected, resulting in poor culture performance. In acidic culture environments, temperature significantly affected the pac expression level and specific PAC activity decreased with an increase in culture temperature. In basic culture environments, cell growth was seriously inhibited though the pac expression level was minimally affected by temperature. Such unusual types of pH and temperature effects on pac expression were never reported for bacterial PACs. The results suggest that culture pH should be precisely controlled for the current host/vector systems being applied on the overproduction of P. rettgeri PAC in E. coli at high temperatures.     

Interaction of hyperthermia and pentamidine in HeLa S-3 cells

Pentamidine is similar to rhodamine-123 in chemical structure and state of electron charge, and rhodamine-123 was previously shown to be a hyperthermic sensitizer under appropriate cell culture conditions. The present experiments were carried out to determine whether pentamidine would potentiate hyperthermic cell killing and, if so, under what cultural conditions. Exposure of HeLa cells to pentamidine (80 microM) up to 4 h was not cytotoxic in culture medium in the presence or absence of glucose at 37 degrees C and pH 7.4. Cells in the glucose-deprived medium became progressively sensitive to killing as temperature and drug concentration were increased. On the other hand, there was a moderate level of enhanced cell killing in the glucose-fed medium at 42 degrees C. The enhanced effects of heat by the drug were most pronounced under alkaline pH of the culture medium. The cell kill under acidic pH was far less than that observed at neutral or alkaline pH; these effects may be a result of decreased cellular uptake of pentamidine. Together with our previous results on rhodamine-123 and glycolytic inhibitors, the present data with pentamidine are consistent with the concept that deprivation of cellular energy increases sensitivity to cell killing by heat.     

Lupin peroxidases. II. Binding of acidic isoperoxidases to cell walls

Extracellular acidic isoperoxidases (EC 1.11.1.7), isolated from both the cell walls and intercellular spaces of lupin ( Lupinus albus L. cv. multolupa) hypocotyls, bound to water-insoluble pectins of wall fragments also isolated from the hypocotyls. The binding was sáturable by increasing the isoenzyme concentration in the assay medium and it was dependent on the pH; neutral pH (6.0–7.0) favoured release, while acidic pH (4.0–5.0) favoured the attachment to the cell wall. Binding of acidic isoperoxidases to wall fractions was correlated with the in vitro acid-induced growth of hypocotyl segments, and both were modulated in the same direction by the Ca²⁺/H⁺ ratio in the incubation media, although the two responses were clearly separated when the Ca²⁺/H⁺ ratio varied. Binding of acidic isoperoxidases of cell walls could operate as a fine control of the activity of these cell wall enzymes, although its physiological role in the cell wall stiffening remains unclear. Some aspects of Ca²⁺ on the control of peroxidase activity at this level are also discussed.     

Enhanced intracellular delivery of quantum dot and adenovirus nanoparticles triggered by acidic pH via surface charge reversal

Quantum dot (QD) and adenovirus (ADV) nanoparticles were surface-modified with graft copolymers that exhibited a charge reversal behavior under acidic condition. Poly(L-lysine) (PLL) was grafted with multiple biotin-PEG chains (biotin-PEG-PLL graft copolymer), and the remaining primary amine groups in the PLL backbone were postmodified using citraconic anhydride, a pH-sensitive primary amine blocker, to generate carboxylate groups. The surfaces of streptavidin-conjugated QDs were modified with citraconylated biotin-PEG-PLL copolymer, producing net negatively charged QD nanoparticles. Under acidic conditions, citraconylated amide linkages were cleaved, resulting in the recovery of positively charged amine groups with subsequent alteration of surface charge values. Intracellular delivery of QD nanoparticles was greatly enhanced in an acidic pH condition due to the surface charge reversal. The surface of avidin-conjugated adenovirus (ADV-Avi) encoding an exogenous green fluorescent protein (GFP) gene was also modified in the same fashion. The expression extent of GFP was significantly increased at more acidic pH than pH 7.4. This study demonstrates that various nanosized drug carriers, imaging agents, and viruses could be surface-engineered to enhance their cellular uptake specifically at a low pH microenvironment like solid tumor tissue.