Inactivation during phagocytosis of leucine aminopeptidase, an ecto-enzyme of polymorphonuclear neutrophils

Changes in enzyme activities of the plasma membrane makers were examined during phagocytosis using guinea-pig polymorphonuclear neutrophils. Incubation of neutrophils with fresh serum-opsonized zymosan particles showed a significant reduction in leucine aminopeptidase activity, whereas 5′-nucleotidase and alkaline phosphodieterase activities remained unchanged. Inactivation of leucine aminopeptidase activity was also observed by exposure of neutrophils to complement-opsonized zymosan particles, but not to non-opsonized zymosan, IgG-coated zymosan or polysterene latex particles. Pretreatment of neutrophils with cytochalasin B, which prevents phagocytosis but not surface binding of particles, provoked inactivation to the same degree as when the cells were allowed to phagocytose the particles. However, the inactivation during phagocytosis was protected by serine protease inhibitors. These findings suggest that loss of leucine aminopeptidase activity from phagocytosing cells may be mediated by certain serine protease inhibitor-sensitive factor(s) which are probably activated by the attachment of an opsonized zymosan particle to a specific membrane receptor, probably the C3b receptor.     

Detection of an Intracellular Protease Inhibitor in Archaea

Proteolytic activity and a subtilisin inhibitor (NSI) were detected in Natrialba magadii cells. The proteolytic activity was due to two different proteases: a ∼90-kDa metallo protease (NMP) produced during exponential growth and a 246-kDa serine protease (NSP) detected in the stationary phase. Both proteases were detected in the cytosolic fraction. NSI activity was maximal during early stages of growth and decreased in the stationary phase. NSI is a 35-kDa thermosensitive protein; it inhibits NSP activity but has no effect on NMP, and it was detected as a soluble or membrane-bound protein depending on the growth phase. Our results suggest that NSI may regulate NSP activity in vivo and that this protease may have a role in stationary phase cells. To our knowledge, this is the first report on the occurrence of protease inhibitors in Archaea. Received: 4 May 2002 / Accepted: 10 July 2002     

One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers.     

Changes in bacterial cells induced by high pressure at subzero temperature

The aim of this study was to examine the effect of pressure treatment at 193 MPa and −20 °C on membrane damage, changes in activity of membrane-bound ATPases and degradation of nucleic acids. The experiments were carried out with three Escherichia coli strains, in the exponential and stationary phases of growth, and differing in sensitivity to pressure. All E. coli strains subjected to pressure in the exponential phase of growth were inactivated by 6 log cycles, independently of the strain, which was accompanied by a total loss of ability to plasmolyse, an increase in irreversible membrane permeability to PI, and a reduction of cellular ATP by more than 80%. After pressure treatment of stationary phase cells, the relationship between the inactivation level and the ability to plasmolyse was not as evident as in the case of exponential phase cells. Pressure treatment of two strains of E. coli K-12 and Ec160/59 in the stationary phase that decreased viability by no more than one log cycle led only to reversible permeabilization of bacterial membranes, while irreversible permeabilization was observed in the pressure sensitive E. coli IBA72 strain phase that was inactivated by 4.6 log cycles. The reduction of ATP and changes in ATPase activity after pressure treatment of tested E. coli strains in the stationary phase of growth depended on the stage of inactivation of the particular strain. Electrophoretic analysis showed degradation of RNA isolated after pressure treatment from cells of all E. coli strains tested in the exponential phase of growth. The changes of RNA induced by pressure were not visible in the case of cells in the stationary phase. The degradation of DNA isolated from pressure treated E. coli strains from the exponential as well as from the stationary phase of growth was not observed.     

Behavior in two-phase aqueous polymer systems of L5178Y mouse leukemic cells : II. The lag and exponential phases of growth

The behavior of lag and exponential growth phase L5178Y mouse leukemic cells under normal and prolonged lag phase conditions with respect to partition in aqueous dextran — polyethylene glycol polymer systems has been studied. ‘Backculture’ of early stationary cells into fresh growth medium is accompanied by a decrease in partition ratio from 0.52 to 0.11. The partition ratio remains depressed for a time considerably longer than the duration of lag phase but rises rapidly and returns to its former value as the cells reach late exponential/early stationary phase. If lag phase is prolonged, the time for which the partition ratio remains depressed is also prolonged. In the exponential phase following a prolonged lag phase, the partition ratio rises at a rate slower than during a normal exponential phase and does not reach the same magnitude for the same position in the cycle. Net negative surface charge as measured by particle microelectrophoresis does not change appreciably throughout the growth cycle. The results suggest that the sequence of events at the cell surface on a populational basis which contribute to the partitioning behavior is possibly predetermined or programmed at the time of transfer into fresh medium. The results further substantiate the technique of aqueous polymer partitioning as being the most sensitive method available for monitoring subtle changes in plasma membrane properties during the cell growth cycle.     

HearNPV在生长对数期与平台期宿主细胞中的复制差异分析

使用实时荧光定量PCR技术对HearNPV在生长对数期和平台期HzAM1细胞的复制差异进行分析。结果表明,HzAM1细胞生长对数期的倍增时间为22 h,生长对数期的细胞以S期细胞为主(48.6%),而平台期细胞中以G2/M期细胞为主(72.6%)。在这两种不同状态的细胞中,病毒的复制主要在感染后60 h内完成,在感染后14~20 h,病毒复制倍增时间分别为1.8 h和1.9 h,几乎没有差别。但是感染生长对数期细胞时,吸附侵入细胞内的BV数量、BV释放的数量、最终的病毒产量以及病毒表达的蛋白产量明显高于被病毒感染的生长平台期细胞。如生长对数期细胞内复制合成的病毒DNA总量的25%装配形成BV病毒粒子出芽释放到细胞外,而对于平台期细胞,病毒DNA仅有13%装配形成BV病毒粒子出芽释放到细胞外。病毒感染两种生长状态的细胞,病毒DNA均从感染后7~8 h开始复制,没有明显差别;而生长对数期细胞从被感染后18~20 h释放子代病毒BV,生长平台期细胞则在感染后22~25 h开始释放病毒BV。在感染后30~60 h,在生长对数期被感染的细胞释放BV的速度约为483 copies/cell/h,而平台期细胞约为100 copies/cell/h。最初吸附侵入到生长对数期细胞内的BV粒子数量明显多于侵入到生长平台期细胞内的BV数量。实验证实,生长对数期与平台期的细胞膜的流动性有很大差别,推测健康细胞表面有活性的病毒受体数量可能决定了侵入细胞内的BV的数量。     

Synthesis of α-amylase and α-glucosidase by membrane bound ribosomes from Bacilluslicheniformis

α-Glucosidase was membrane bound during exponential growth of $\text{Bacillus}\text{licheniformis}$ but was released into the medium during stationary phase. It could be partially removed from exponential phase cells by washing with NaCl (0.5 M). α-Amylase was exclusively extracellular and could not be detected in cells. Polysomes were prepared from exponential phase cells and separated into membrane bound and soluble fractions. $\text{In}\text{vitro}$ chain completion and immunoprecipitation showed that α-glucosidase and α-amylase were synthesized by membrane bound and not by soluble ribosomes.     

Variations in the activity of fatty ACYL-CoA desaturases and electron-transport components in Tetrahymena microsomes with temperature and age of culture

1. 1.|Alterations in the fatty acid composition of microsomes were most marked in the exponential phase of both 39.5- or 15°C- grown Tetrahymena pyriformis NT-1.

2. 2.|Activities of palmitoyl-CoA and stearoyl-CoA desaturases were lower in 15°C cells than in 39.5°C cells, while the activity of oleoyl-CoA desaturase was higher in 15°C cells.

3. 3.|Activities of the terminal component of the desaturation system as well as all three desaturases (palmitoyl-CoA, stearoyl-CoA, oleoyl-CoA) were higher in the exponential phase than in the stationary phase for cells grown at both temperatures.

4. 4.|NAD(P)H-cytochrome c reductase activity and cytochrome b₅ content were reduced whereas NADH-ferricyanide reductase activity was increased in the stationary phase at both 39.5 and 15°C.

Relationship between membrane damage and cell death in pressure-treated Escherichia coli cells: differences between exponential- and stationary-phase cells and variation among strains

The relationship between membrane damage and loss of viability following pressure treatment was examined in Escherichia coli strains C9490, H1071, and NCTC 8003. These strains showed high, medium, and low resistance to pressure, respectively, in stationary phase but similar resistance to pressure in exponential phase. Loss of membrane integrity was measured as loss of osmotic responsiveness or as increased uptake of the fluorescent dye propidium iodide. In exponential-phase cells, loss of viability was correlated with a permanent loss of membrane integrity in all strains, whereas in stationary-phase cells, a more complicated picture emerged in which cell membranes became leaky during pressure treatment but resealed to a greater or lesser extent following decompression. Strain H1071 displayed a very unusual pressure response in stationary phase in which survival decreased to a minimum at 300 MPa but then increased at 400 to 500 MPa before decreasing again. Membranes were unable to reseal after treatment at 300 MPa but could do so after treatment at higher pressures. Membrane damage in this strain was thus typical of exponential-phase cells under low-pressure conditions but of stationary-phase cells under higher-pressure conditions. Heat shock treatment of strain H1071 cells increased pressure resistance under low-pressure conditions and also allowed membrane damage to reseal. Growth in the presence of IPTG (isopropyl-beta-D-thiogalactopyranoside) increased resistance under high-pressure conditions. The mechanisms of inactivation may thus differ at high and low pressures. These studies support the view that membrane damage is an important event in the inactivation of bacteria by high pressure, but the nature of membrane damage and its relation to cell death may differ between species and phases of growth.     

Effect of subchronic treatment with mercury chloride on NTPDase, 5′-nucleotidase and acetylcholinesterase from cerebral cortex of rats

The aim of the present investigation was to evaluate the effect of a subchronic treatment (30 days/30 doses) with subcutaneous injections (0.1 mg/kg) of HgCl₂ on NTPDase (E.C. 3.6.1.5), 5′-nucleotidase (E.C. 3.1.3.5) and acetylcholinesterase (AChE, E.C. 3.1.1.7) activities in brain from adult rats. NTPDase and 5′-nucleotidase were measured in cortical synaptosomal fraction and AChE was measured in the homogenate of cerebral cortex and hippocampus. After the subchronic treatment (30 days), NTPDase activity was enhanced approximately 35% (p < 0.05) with ATP and ADP as substrates and no difference was observed in 5′-nucleotidase activity (AMP hydrolysis). In addition, AChE activity was enhanced in the cerebral cortex (22%, p < 0.05) and hippocampus (26%, p < 0.05) after the subchronic treatment. Mercury deposited in brain was measured by cold vapor (atomic absorption spectrometry) and no difference between the control and the subchronically treated group was observed. Here we showed for the first time that exposure to low levels of Hg²⁺, which resembles occupational exposure to low levels of mercury, caused a marked increase in NTPDase and AChE activities. The relationship of these alterations with the neurotoxicity of inorganic mercury deserves further studies.     

Regulation of activity of an ATP-dependent protease, Clp, by the amount of a subunit, ClpA, in the growth of Escherichia coli cells

The activity of an ATP-dependent protease, Clp, was examined in Escherichia coli SG1110 (lon-) in various growth phases. The ATP-dependent proteolytic activity (Clp activity) in a crude extract of the cells changed with the growth phase. Cells in the early exponential growth phase showed the lowest activity, but then the activity increased dramatically with cell growth. The highest Clp activity was found in the cells in the late exponential and early stationary phases, however, the activity returned to the original level on prolonged culturing. These changes in Clp activity were closely correlated to the amount of one of the components of Clp, Clp A, which was quantitated immunochemically with antibodies against the Clp A protein. However, the amount of the other component of Clp, Clp P, did not change with the growth phase. These results suggest that the activity of Clp in the cells is regulated by the amount of Clp A in various growth phases. We next examined the effect of the cellular ATP level on Clp activity, because ATP is a cofactor for Clp protease in vitro. The addition of dinitrophenol (DNP) and sodium azide reduced the intracellular concentration of ATP, but had no effect on the Clp activity or the level of the Clp A protein when these drugs were added to the culture at the stationary phase. On the other hand, these drugs elevated both the Clp activity and the Clp A amount in exponentially growing cells, whose cellular ATP level was also reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and release of proteases byBacteroides fragilis

Protease production byBacteroides fragilis ATCC 25285 was determined in batch and continuous cultures. During exponential growth in batch culture, the majority of proteolysis was cell associated. However, as the bacteria reached stationary phase, most of the intracellular proteases were released into the culture medium. Measurements of alkaline phosphatase and -galactosidase, which are respectively periplasmic and cytoplasmic marker enzymes inB. fragilis, showed that secretion of proteases in the stationary phase was a discrete event and was not associated with a general release of cytoplasmic contents. When the bacterium was grown in continuous culture, cell-associated protease activity increased concomitantly with dilution rate (D=0.03–0.23/h). The ratio of intracellular to whole cell protease activity also increased with growth rate (11 at D=0.03/h; 11.7 at D=0.23/h). Extracellular protease activity was detected only in trace amounts in continuous cultures at the lowest dilution rate. Determinations of the distribution of extracellular protease activity in batch culture after 48 h incubation showed that the majority of proteolysis (ca. 90%) was soluble. Nevertheless, a proportion was associated with particulate fractions, which had high specific activities.     

Ion-pair reversed-phase high-performance liquid chromatography of adenine nucleotides and nucleoside using triethylamine as a counterion

An isocratic HPLC method for the simple and selective determination of adenine nucleoside and nucleotides has been developed. The separation is achieved at room temperature by reversed-phase chromatography (Shiseido, Capcell Pak C18). A mixture of 0.1 M triethylamine (TEA) phosphate buffer and methanol (95:5, v/v) is used as a standard eluent. Influence of pH and concentrations of organic modifiers and TEA ion on capacity factors of adenine compounds has been investigated. It has been also found that the TEA ion in the eluent is adsorbed onto the reversed-phase surface. The results clearly demonstrate that ion-pair formation with TEA ion occurs probably both in the mobile phase and on the stationary phase and governs the retention of adenine and nucleotides in the present system. The HPLC system is applied to the analysis of adenine nucleotides formed as intermediates in the synthesis of 3′-phosphoadenosine 5′-phosphosulphate (PAPS) and to the assays of ATPases and 5′-nucleotidase activities in rat liver plasma membrane. This method is a new type of ion-pair reversed-phase HPLC system and is suitable for the separation of highly polar organic anions, especially for adenine nucleotides.     

Detection of quorum sensing signals in the haloalkaliphilic archaeon Natronococcus occultus

Bacteria communicate at high cell density through quorum sensing, however, there are no reports about this mechanism in archaea. The archaeon Natronococcus occultus produces an extracellular protease at the end of growth. Early production of protease activity was observed when a low density culture was incubated with late exponential conditioned medium suggesting the presence of factor(s) inducing this activity. Conditioned medium and ethyl acetate extracts corresponding to the transition from exponential to stationary phase showed a positive signal in Agrobacterium biosensor. We report the detection of potential autoinducer molecules of the acylated homoserine lactone type in the archaeon N. occultus. These molecules may be responsible for the production/activation of extracellular protease.     

Heparin and chondroitin sulfate inhibit adenine nucleotide hydrolysis in liver and kidney membrane enriched fractions

The inhibition of adenine nucleotide hydrolysis by heparin and chondroitin sulfate (sulfated polysaccharides) was studied in membrane preparations from liver and kidney of adult rats. Hydrolysis was measured by the activity of NTPDase and 5′-nucleotidase. The inhibition of NTPDase by heparin was observed at three different pH values (6.0, 8.0 and 10.0). In liver, the maximal inhibition observed for ATP and ADP hydrolysis was about 80% at pH 8.0 and 70% at pH 6.0 and 10.0. Similarly to the effect observed in liver, heparin caused inhibition of ATP and ADP hydrolysis that reached a maximum of 70% in kidney (pH 8.0). Na⁺, K⁺ and Rb⁺ changed the inhibitory potency of heparin, suggesting that its effects may be related to charge interaction. In addition to heparin, chondroitin sulfate also caused a dose-dependent inhibition in liver and kidney membranes. The maximal inhibition observed for ATP and ADP hydrolysis was about 60 and 50%, respectively. In addition, the hepatic and renal activity of 5′-nucleotidase was inhibited by heparin and chondroitin sulfate, except for kidney membranes where chondroitin sulfate did not alter AMP hydrolysis. On this basis, the findings indicate that glycosaminoglycans have a potential role as inhibitors of adenine nucleotide hydrolysis on the surface of liver and kidney cell membranes in vitro.     

Dolichyl phosphate phosphatase from Tetrahymena pyriformis.

A soluble dolichyl phosphate phosphatase from Tetrahymena pyriformis was purified about 68-fold. The enzyme appeared to be specific for dolichyl phosphate and existed in two interrelated forms, one of mol.wt. about 500000 and the other of mol.wt. about 63000. The enzyme was strongly inhibited by 5 mM-Mn2+ and was strongly stimulated by Mg2+. Tetrahymena in the exponential growth phase contained more of this enzymic activity than cells in stationary or lag phase. The dolichyl phosphate phosphatase may be loosely bound to mitochondrial membranes. Two roles proposed for this enzyme are (1) that of releasing dolichol from its phosphorylated biosynthetic form for its use in the cell as unesterified dolichol or dolichyl ester and/or (2) that of regulation of synthesis of glycoproteins or some other glycosylated compound.     

The measurement of cyclic GMP and cyclic AMP phosphodiesterases

Methods are described for measuring phosphodiesterases for cGMP and cAMP in the range of activity yielding 10⁻¹² to 10⁻⁸ mol of product. The 5′-GMP formed is measured by conversion to GDP with guanylate kinase. Amounts of GDP greater than 10⁻¹⁰ mol are measured directly with an enzyme system which results in stoichiometric oxidation of NADH. This is either determined by the decrease in fluorescence or the excess NADH is destroyed with acid and the NAD⁺ measured by its fluorescence in strong NaOH. With smaller amounts of GDP, sensitivity is amplified 1000-fold with the succinic thiokinase-pyruvate kinase cycle. In the case of cAMP diesterase, larger amounts of 5′-AMP are measured in the same way as 5′-GMP, except that adenylate kinase is substituted for guanylate kinase. With smaller amounts, the 5′-AMP is converted to ATP, and sensitivity is amplified with the adenylate kinase-pyruvate kinase cycle. As little as 20 ng dry weight of average brain is sufficient for accurate assay of the diesterase activity toward either cAMP or cGMP. When there is danger of significant destruction of AMP or GMP by tissue 5′-nucleotidase, this is prevented by adding GMP to the cAMP reagent, AMP to the cGMP reagent, or 5′-UMP to either reagent.     

5′-nucleotidase activity in cultured cell lines. Effect of different assay conditions and correlation with cell proliferation

Summary The 5′-AMPase activity of the ectoenzyme 5′-nucleotidase has been measured in a variety of cell lines, using intact cells. Human cell types showed two orders of magnitude higher enzyme activity than mouse cell lines. The ectoenzyme is inhibited by adenosine 5′-(α,β-methylene) diphosphate and Concanavalin A. A different extent of 5′-nucleotidase lectin inhibition was observed in the studied cell lines, suggesting that the corresponding ectoenzymes are glycoproteins with a different type or degree, or both, of glycosylation. The 5′-nucleotidase activity increased during subculture and decreased after cell transformation. Generally, the 5′-nucleotidase activity was two-to five-fold higher in monolayer than in suspension cell culture. A relation between cell growth and 5′-AMPase activity was also observed. Enzyme activity increased at the end of the lag phase (glioblastoma cells) or during the exponential phase (the other two cell lines). After confluence, the activity decreased to the initial or even lower range of activity. Observed activity variations with cell proliferation correlate with modifications of 5′-AMPase activity during subculture. This work was supported by grant no. PR84-0359 from the Comisión Asesora de Investigación Científica y Técnica (Spain).     

Inactivation of cell-associated fructosyltransferase in Streptococcus salivarius.

In stationary phase, 95% of the fructosyltransferase (FTase) activity of Streptococcus salivarius ATCC 25975 was found associated with the cells. Within the first 15 min after inoculation into fresh medium, the specific activity of cell-associated FTase decreased by 92% of its initial value. After this period of initial loss, the enzyme was synthesized during exponential growth until a maximum level equivalent to that present before inoculation was obtained. The inactivation of FTase was also demonstrated in a nongrowing system. Washed cell suspensions incubated at 37 degrees C in 200 mM potassium phosphate buffer (pH 6.5) containing 10 microM Cu2+ lost 80 to 95% of their FRase activity after 30 min. This loss could be prevented by the addition of histidine, cysteine, or Ca2+ to the suspension mixture. A factor(s) essential for the inactivation of cell-associated FTase could itself be preferentially inactivated by heating cells at 40 degrees C for periods of up to 3 h, or by storage of cells at 0 to 4 degrees C for several days in a low-ionic-strength, low-pH, potassium phosphate buffer. Treatment of cells with the N-acetylmuramidase enzyme M-1, in the presence of 0.5 M melezitose, resulted in the release of FTase from the cell. The released enzyme was recovered in the supernatant fraction after centrifugation at 160,000 x g for 90 min. Comparison of solubilized active and inactivated FTase preparations by polyacrylamide gel electrophoresis demonstrated that the inactivation of cell-associated FTase activity was associated with the loss of specific protein bands.