Analysis of survival rates and cellular fatty acid profiles of Listeria monocytogenes treated with supercritical carbon dioxide under the influence of cosolvents

In the present study, we identified several process variables that significantly affect the efficiency of supercritical carbon dioxide inactivation of the food-borne pathogen Listeria monocytogenes. Treatment with SC-CO(2) completely disabled the colony-forming activity of the cells (8-log reduction) within specific treatment time (10-50 min), pressure (80-150 bar), and temperature ranges (35-45 degrees C). Microorganism inactivation rates increased proportionally with pressure and temperature, but the inactivation rate decreased significantly when cells were suspended in phosphate-buffered saline rather than in physiological saline. Additionally, when the microbial cell suspension was 80-100% (w/w) of water, the SC-CO(2)-mediated reduction in CFU ml(-1) was 4-8 log higher at the same treatment conditions than in typical cell suspensions (a water content of 800-4000% [w/w]) or dry preparations that had only 2-10% (w/w) of water. The addition of a fatty acid, oleic acid, decreased the effectiveness of the microbial inactivation by SC-CO(2), but the addition of a surfactant, sucrose monolaurate, increased the effectiveness. Therefore, cosolvents for SC-CO(2), including water, a fatty acid, and a surfactant in this study, were found to greatly influence on the inactivation effectiveness. The extraction of cellular substances, such as nucleic acid- and protein-like materials and fatty acids, was monitored by spectrophotometry and GC/MS and increased with SC-CO(2) treatment time. Additionally, using scanning and transmission electron microscopies, we investigated morphological changes in the SC-CO(2)-treated cells. The effects of the variables we have described herein represent a significant contribution to our current knowledge of this method of inactivating food-borne pathogens.     

Ethanol addition on inactivation of Saccharomyces pastorianus by a two‐stage system with low‐pressure carbon dioxide microbubbles can accelerate the cell membrane injury

The effect of ethanol on the inactivation of Saccharomyces pastorianus by a two‐stage system with low‐pressure carbon dioxide microbubbles (two‐stage MBCO₂) was investigated. Zero and >5 log reductions of S. pastorianus populations suspended in physiological saline (PS) containing 0% and 10% ethanol, respectively, occurred by the two‐stage MBCO₂ at a mixing vessel pressure of 1 MPa and a heating coil temperature of 40°C. Conversely, the detected number of surviving S. pastorianus cells in PS containing 5% ethanol was higher in yeast and mold agar (YMA, an optimum agar) than YMA with 2.5% sodium chloride, followed by yeast nitrogen base agar (YNBA, a minimum agar). The fluorescence polarization of S. pastorianus in PS containing 5% and 10% ethanol increased similarly with exposure time in the heating coil of two‐stage MBCO₂ and was correlated with the surviving cell number measured in YNBA. The intracellular pH (pH_in) of S. pastorianus in PS containing 5% ethanol decreased linearly with exposure time in the heating coil of two‐stage MBCO₂. Also, the pH_in‐lowering of S. pastorianus in PS containing 10% ethanol was drastically caused by two‐stage MBCO₂ at 1 min exposure time in the heating coil but then stayed constant until 5 min, agreeing with the inactivation efficiency. Therefore, ethanol in S. pastorianus suspension was suggested to accelerate the cell membrane injury caused by two‐stage MBCO₂. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:282–286, 2018     

High pressure homogenization inactivation of Escherichia coli and Staphylococcus aureus in phosphate buffered saline,milk and apple juice

High pressure homogenization (HPH) offers new opportunities for food pasteurization/sterilization. Escherichia coli and Staphylococcus aureus suspended in phosphate buffered saline (PBS) buffer, milk and apple juice at initial concentration of ~10⁶ log₁₀ CFU per ml were subjected to HPH treatments up to 200 MPa with inlet temperatures at 4–40°C. After HPH at 200 MPa with the inlet temperature at 40°C, the count of E. coli suspended in PBS, milk and apple juice reduced by 3·42, 3·67 and 3·19 log₁₀ CFU per ml respectively while the count of S. aureus decreased by 2·21, 1·02 and 2·33 log₁₀ CFU per ml respectively suggesting that S. aureus was more resistant. The inactivation data were well fitted by the polynomial equation. Milk could provide a protective effect for S. aureus against HPH. After HPH at 200 MPa with the inlet temperature at 20°C, the cell structure of E. coli was destroyed, while no obvious damages were found for S. aureus.     

The effect of high hydrostatic pressure on Listeria monocytogenes in phosphate-buffered saline and model food systems

Three strains of Listeria monocytogenes (NCTC 11994, a poultry isolate and the Scott A strain) were exposed to a range of pressures (300, 350, 375, 400 and 450 MPa) in 10 mmol l⁻¹ phosphate-buffered saline (PBS) at pH 7·0 for up to 30 min at ambient temperature. Generally, increasing the magnitude and duration of compression resulted in increasing levels of inactivation, although the inactivation kinetics varied depending on the strain and pressure applied. The three strains also exhibited a wide variation in their resistance to high pressure. The resistance of the three strains to high pressure (375 MPa) was also assessed in a series of model food systems containing one of each of the three main food constituents: protein (1, 2, 5 and 8% w/v bovine serum albumin in PBS), carbohydrate (1, 2, 5 and 10% w/v glucose in PBS) and lipid (olive oil (30% v/v) in PBS emulsion). Overall, increasing the concentrations of bovine serum albumin (BSA) and glucose in the suspending medium resulted in decreasing levels of inactivation of all three strains; however, the minimum concentration of BSA and glucose required to increase survival to a level greater than that observed in PBS alone varied depending on the strain and on the duration of the treatment. The survival of all three strains was greater in the olive oil/PBS emulsion than in PBS alone at all treatment times.     

The reaction of chemical probes with the erythrocyte membrane.

Trinitrobenzenesulfonate (TNBS), fluorodinitrobenzene (FDNB) and suberimidate have been reacted with intact human erythrocytes. TNBS does not penetrate the cell membrane significantly at 23 degrees C in bicarbonate-NaCl buffer, pH 8.6, as estimated by the labeling of the N-terminal valine of hemoglobin. Hence, under these conditions it can be used as a vectorial probe. However, at 37 degrees C, especially in phosphate buffer, at pH 8.6, TNBS does penetrate the cell membrane. FDNB and suberimidate both penetrate the erythrocyte membrane. The time course reaction of TNBS with intact erythrocytes over a 24-hr period at 23 degrees C is complex and shows transition zones for both membrane phosphatidylethanolamine (PE) and membrane proteins. No significant cell lysis occurs up to 10 hr. The fraction of total PE or phosphatidylserine (PS) which reacts with TNBS by this time period can be considered to be located on the outer surface of the cell membrane. Under these conditions it can be located on the outer surface of the cell membrane. Under these conditions it can be shown that 10 to 20% of the total PE and no PS is located on the outer surface of the membrane and hence these amino phospholipids are asymmetrically arranged. The pH gradient between the inside and outside of the cell in our system is 0.4 pH units. Nigericin has no effect on the extent of labeling of PE or PS by TNBS. Isotonic sucrose gives a slight enhancement of the labeling of PE by TNBS. Hence, the inability of PE and PS to react with the TNBS is considered not due to the inside of the cell having a lower pH. The extent of reaction of TNBS with PE is not influenced by changing the osmolarity of the medium or by treatment of cells with pronase, trypsin, phospholipase A or phospholipase D. However, bovine serum albumin (BSA) does protect some of the PE molecules from reacting with TNBS. Cels treated with suberimidate were suspended in either isotonic NaCl or in distilled water. In both cases the suberimidate-treated cells became refractory to hypotonic lysis. Pretreatment of cells with TNBS did not prevent them from interacting with suberimidate and becoming refractory to lysis. However, pretreatment of cells with the penetrating probe FDNB abolished the suberimidate effect. Electron-microscopic analysis of the cells showed a continuous membrane in the case of cells suspended in isotonic saline. The cells suspended in water did not lyse but their membranes had many large holes, sufficient to let the hemoglobin leak out. Since the hemoglobin did not leak out we know that the hemoglobin is cross-linked into a large supramolecular aggregate.     

Radiation-induced generation of chlorine derivatives in N2O-saturated phosphate buffered saline: toxic effects on Escherichia coli cells.

The radiolysis of aqueous chloride solutions has been investigated using pulse and steady-state methods. We have found a correlation between the yields of Cl2- and HOCl formed in pulse-irradiated N2O-saturated solutions. The yields increased with the increasing concentrations of Cl- and phosphate. Phosphate enhanced the yield of Cl2- in neutral solutions because of a proton transfer from H2PO4- to HOCl- with a rate constant of (2.6 +/- 0.5) x 10(8) M-1s-1. HOCl could not be detected in pulse-irradiated He or air-saturated, phosphate-buffered saline (PBS) solutions or in gamma-irradiated N2O, He, or air-saturated PBS solutions. The results are discussed in light of previously suggested mechanisms for the formation and decay of Cl2-. Pulse-irradiated N2O-saturated PBS solutions have a lethal effect on Escherichia coli cells, which is proportional to the amount of HOCl in the solutions. Gamma-irradiation of cells in N2O-saturated PBS solution also raises the radiosensitivity of the cells, although HOCl does not accumulate in this system. The effects of the radiation-induced toxic products on E. coli cells are similar to the effects of NaOCl. The cell membrane is probably the site of physiological injury induced by the radiation products.     

Insights into virus inactivation by polysorbate 80 in the absence of solvent

Triton X-100 has long been used either alone or in combination with solvent to inactivate enveloped viruses in biopharmaceutical manufacturing. However, European Chemicals Agency (ECHA) officially placed Triton X-100 on the Annex XIV authorization list in 2017 because 4-(1,1,3,3-tetramethylbutyl) phenol, a degradation product of Triton X-100, is of harmful endocrine disrupting activities. As a result, any use of Triton X-100 in the European Economic Area would require an ECHA issued authorization after the sunset date of January 4, 2021. In search of possible replacements for Triton X-100, we discovered that polysorbate 80 (PS80) in absence of any solvents was able to effectively inactive enveloped viruses such as xenotropic murine leukemia virus and pseudorabies virus with comparable efficacy as measured by log reduction factors. Interestingly, PS80 did not show any virucidal activities in phosphate buffered saline (PBS) while achieving robust virus inactivation in cell-free Chinese hamster ovary (CHO) bioreactor harvests. This intriguing observation led us to speculate that virus inactivation by PS80 involved components in the cell-free CHO bioreactor harvests that were absent in PBS. Specifically, we hypothesized that esterase and/or lipases in the cell-free bioreactor harvests hydrolyzed PS80 to yield oleic acid, a known potent virucidal agent, which in turn inactivated viruses. This theory was confirmed using purified recombinant lysosomal phospholipase A2 isomer (rLPLA2) in PBS. Subsequent characterization work has indicated that virus inactivation by PS80 is effective and robust within temperature and concentration ranges comparable to those of Triton X-100. Similar to Triton X-100, virus inactivation by PS80 is dually dependent on treatment time and temperature. Unlike Triton X-100, PS80 inactivation does not correlate with concentrations in a simple manner. Additionally, we have demonstrated that PS20 exhibits similar virus inactivation activities as PS80. Based on the findings described in the current work, we believe that PS80 is potentially a viable replacement for Triton X-100 and can be used in manufacturing processes for wide spectrum of biopharmaceuticals to achieve desirable virus clearance. Finally, the advantages and disadvantages of using PS80 for virus inactivation are discussed in the contexts of GMP manufacturing.     

Chromosomal location and expression of the structural gene for major outer membrane protein Ia of Escherichia coli K-12 and of the homologous gene of Salmonella typhimurium.

The gene determining the structure of a major outer membrane protein of Escherichia coli, protein Ia, has been located between serC and pyrD, at the min 21 region of the linkage map. This is based on the isolation and characterization of E. coli-Salmonella typhimurium intergeneric hybrids as well as analyses of a mutation (ompF2) affecting the formation of protein Ia. When the serC region of the S. typhimurium chromosome was transduced by phage P1 into E. coli, two classes of transductants were obtained; one produced protein Ia like the parental strain of E. coli, whereas the other produced not protein Ia but a pair of outer membrane proteins structurally related to 35K protein, one of the major outer membrane proteins of S. typhimurium. Furthermore, a strain of S. typhimurium harboring an F' plasmid which carries the ompF region of the E. coli chromosome was found to produce a protein indistinguishable from protein Ia, beside the outer membrane proteins characteristic to the parental Salmonella strain. These results suggest that the structural genes for protein Ia (E. coli) and for 35K protein (S. typhimurium) are homologous to each other and are located at the ompF region of the respective chromosome. The bearing of these findings on the genetic control of protein Ia formation is discussed.     

The reaction of chemical probes with the erythrocyte membrane

Summary Trinitrobenzenesulfonate (TNBS), fluorodinitrobenzene (FDNB) and suberimidate have been reacted with intact human erythrocytes. TNBS does not penetrate the cell membrane significantly at 23 °C in bicarbonate-NaCl buffer, pH 8.6, as estimated by the labeling of the N-terminal valine of hemoglobin. Hence, under these conditions it can be used as a vectorial probe. However, at 37 °C, especially in phosphate buffer, at pH 8.6, TNBS does penetrate the cell membrane. FDNB and suberimidate both penetrate the erythrocyte membrane. The time course reaction of TNBS with intact erythrocytes over a 24-hr period at 23 °C is complex and shows transition zones for both membrane phosphatidylethanolamine (PE) and membrane proteins. No significant cell lysis occurs up to 10 hr. The fraction of total PE or phosphatidylserine (PS) which reacts with TNBS by this time period can be considered to be located on the outer surface of the cell membrane. Under these conditions it can be shown that 10 to 20% of the total PE and no PS is located on the outer surface of the membrane and hence these amino phospholipids are asymmetrically arranged. The pH gradient between the inside and outside of the cell in our system is 0.4 pH units. Nigericin has no effect on the extent of labeling of PE or PS by TNBS. Isotonic sucrose gives a slight enhancement of the labeling of PE by TNBS. Hence, the inability of PE and PS to react with the TNBS is considered not due to the inside of the cell having a lower pH. The extent of reaction of TNBS with PE is not influenced by changing the osmolarity of the medium or by treatment of cells with pronase, trypsin, phospholipase A or phospholipase D. However, bovine serum albumin (BSA) does protect some of the PE molecules from reacting with TNBS.Cells treated with suberimidate were suspended in either isotonic NaCl or in distilled water. In both cases the suberimidate-treated cells became refractory to hypotonic lysis. Pretreatment of cells with TNBS did not prevent them from interacting with suberimidate and becoming refractory to lysis. However, pretreatment of cells with the penetrating probe FDNB abolished the suberimidate, effect. Electron-microscopic analysis of the cells showed a continuous membrane in the case of cells suspended in isotonic saline. The cells suspended in water did not lyse but their membranes had many large holes, sufficient to let the hemoglobin leak out. Since the hemoglobin did not leak out we know that the hemoglobin is cross-linked into a large supramolecular aggregate.     

Further characterization of the effects of alpha-1-acid glycoprotein on the passage of human erythrocytes through micropores

Effects of human alpha-1-acid glycoprotein (AG) on the passage of human red blood cell(s) (RBC) through membrane filters with micropores were examined in vitro. RBCs, with a mean major diameter of 7.2 micron, that had been suspended at 1% in physiological phosphate-buffered saline (PBS), were filtered through membrane filters of various pore diameters under positive pressure. The percentages of cells that passed through the micropores and of cells hemolyzed during filtration were determined. RBCs suspended in PBS did not pass through micropores that had an average pore diameter of 3 micron; instead hemolysis took place. Neither temperature nor applied pressure affected cell passage; but when AG at 0.1 mg/ml or above was added to an RBC-suspension, it promoted cell passage through the 3 micron micropores and reduced the degree of hemolysis. The effects of AG were dose dependent up to a concentration of 0.5 mg/ml. The addition of AG to an RBC-suspension that contained 90% human serum had the same additive effects. Washing AG-treated RBCs with normal saline produced a marked decrease in cell passage through the 3 micron pores. Fluorescence antibody staining revealed that the exogenous AG was localized on the membrane surface of the RBCs. Our results suggest that the AG bound to the surface of the RBCs acts as a lubricant between the RBCs and the wall of the micropore; this would facilitate RBC-passage through the micropores.     

Formation of cross-linking between photosystem 2 proteins during irradiation of thylakoid membranes at high temperature

Irradiation of thylakoid membranes at 40 °C resulted in complete inhibition of photosystem (PS) 2 activity measured as 2,6-dichlorophenol indophenol (DCIP) photoreduction either in the absence or presence of 1,5-diphenylcarbazide (DPC). Concomitant with the inactivation of PS2 activity, several thylakoid proteins were lost and high molecular mass cross-linking products appeared that cross-reacted with antibodies against proteins of PS2 but not with antibodies against proteins of other three complexes PS1, ATP synthase, and cytochrome b₆f. Irradiation of thylakoid membranes suspended in buffer of basic pH or high concentration of Tris at 25 °C resulted in the formation of cross-linking products similar to those in thylakoid membranes irradiated at 40 °C. Presence of radical scavengers and DPC during the high temperature treatment prevented the formation of cross-linking products. These results suggest the involvement of oxygen evolving co mplex (OEC) in the formation of cross-linking between PS2 proteins in thylakoid membrane irradiated at high temperature. This revised version was published online in September 2006 with corrections to the Cover Date.     

鼠伤寒沙门菌外膜蛋白与耐药性关系的分析

目的分析鼠伤寒沙门菌外膜蛋白(OMP)与耐药性的关系。方法用消除剂丫啶橙消除耐药性,盲传测其遗传稳定性,采用超声波物理裂解法制备鼠伤寒沙门菌外膜蛋白标本,用变性聚丙烯酰胺凝胶电泳(SDA-PAGE)检测外膜蛋白,用紫外分光光度计测其吸光值,计算浓度。结果抗性消除表型能稳定遗传,耐药鼠伤寒沙门菌与敏感鼠伤寒沙门菌都含有6条主要的外膜蛋白条带,两者相比,发现耐药菌的外膜蛋白在约57、53、30 kDa处减弱或缺失,总的蛋白浓度也低于后者。结论鼠伤寒沙门菌耐药性与外膜蛋白的减弱或缺失有关。     

Size heterogeneity of Salmonella typhimurium lipopolysaccharides in outer membranes and culture supernatant membrane fragments.

Enterobacteriaceae cells growing in liquid media shed fragments of their outer membranes. These fragments, which may constitute a biologically important form of gram-negative bacterial endotoxin, have been reported to contain proteins, phospholipids, and lipopolysaccharides (LPS). In this study we compared the sizes of LPS molecules in shed membrane fragments and outer membranes from cells growing in broth cultures. Using conditional mutants of Salmonella typhimurium which incorporate specific sugars into LPS, we analyzed radiolabeled LPS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique revealed that S. typhimurium LPS are more heterogeneous than previously known; molecules possessing from 0 to more than 30 O-chain repeat units were identified in outer membranes, supernatant fragments, and purified LPS. The size distributions of LPS molecules in outer membranes and supernatant fragments were similar; supernatant fragments appeared to be slightly enriched in molecules with long O-polysaccharide chains. Our results indicate the LPS molecules of many sizes are synthesized, translocated to outer membranes, and released into culture supernatants. Since the hydrophilic O-polysaccharides extend from bacterial surfaces into the aqueous environment, our findings suggest that the cell surface topography of this bacterium may be very irregular. We also speculate that heterogeneity in the degree of polymerization of O-antigenic side chains may influence the interactions of the toxic moiety of LPS (lipid A) with host constituents.     

Mathematical modelling of lipid transbilayer movement in the human erythrocyte plasma membrane

A model is presented to simulate transverse lipid movement in the human erythrocyte membrane. The model is based on a system of differential equations describing the time-dependence of phospholipid redistribution and the steady state distribution between the inner and outer membrane monolayer. It takes into account several mechanisms of translocation: (i) ATP-dependent transport via the aminophospholipid translocase; (ii) protein-mediated facilitated and (iii) carrier independent transbilayer diffusion. A reasonable modelling of the known lipid asymmetry could only be achieved by introducing mechanism (iii). We have called this pathway the compensatory flux, which is proportional to the gradient of phospholipids between both membrane leaflets. Using realistic model parameters, the model allows the calculation of the transbilayer motion and distribution of endogenous phospholipids of the human erythrocyte membrane for several biologically relevant conditions. Moreover, the model can also be applied to experiments usually performed to assess phospholipid redistribution in biological membranes. Thus, it is possible to simulate transbilayer motion of exogenously added phospholipid analogues in erythrocyte membranes. Those experiments have been carried out here in parallel using spin labeled lipid analogues. The general application of this model to other membrane systems is outlined.Abbreviations PBS phosphate buffered saline - DFP diisopropyl fluorophosphate - ESR electron spin resonance - RBC red blood cells - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - SM sphingomyelin - (0,2)PC 1-palmitoyl-2(4doxylpentanoyl)-PC - (0,2)PE 1-palmitoyl-2(4-doxylpentanoyl)-PE - (0,2) PS 1-palmitoyl-2(4-doxylpentanoyl)-PS     

Thermal inactivation and stabilization of lysozyme substrate-- Micrococcus lysodeicticus cells

Heat inactivation of the acetonic powder of Micrococcus lysodeicticus cells suspended in phosphate buffer pH 6.2 was quantitatively characterized in the temperature range from 34 to 52 degrees. The total value of the rate constant for heat inactivation of the cells equals 2.88 X 10(8) exp(-18360/RT) sec-1. The activation parameters of the process at 34 degrees are the following: delta H* = 17.7 kcal/mole; delta S* = 21.8 E. U.; delta F* = 24.4 kcal/mole. The effect of ethylene glycol, mannitol, dextran, polyvinyl alcohol (PVA) and polyethylene glycols with different molecular weights on the lysis rate and cell stability was studied. Polyvinyl alcohol was found to be the most effective stabilizer. At concentrations of about 10(-5) it enhances the thermostability of the cells threefold.     

The sugar-specific outer membrane channel ScrY contains functional characteristics of general diffusion pores and substrate-specific porins

Escherichia coli K-12 strain PS1-28-37 carries the multicopy plasmid pPSO28-37 containing a DNA fragment coding for two of the proteins that enable bacteria to utilize sucrose as sole carbon source. One of the different gene products of the plasmid is the outer membrane protein, ScrY. This protein was isolated and purified by chromatography across a gel filtration column. Reconstitution experiments with lipid bilayer membrane demonstrated that ScrY formed ion-permeable channels with properties very similar to those of general diffusion pores of enteric bacteria. The presence of sugars in the aqueous phase led to a dose-dependent block of ion transport through the channel, like the situation found with LamB (maltoporin) of Escherichia coli and Salmonella typhimurium. The binding constants of a variety of different sugars were determined. The stability constant for malto-oligosaccharide binding increased with increasing numbers of glucose residues. Disaccharides generally had a larger binding constant than monosaccharides. The binding of different sugars to ScrY and LamB of E. coli is discussed with respect to the kinetics of sugar movement through the channel.     

Effect of rat polymorphonuclear leukocyte granule components on the growth and survival of Pseudomonas aeruginosa and Salmonella typhimurium

Granule contents from rat polymorphonuclear neutrophils were prepared by extraction with 0.2 M acetate buffer (pH 4.0), dialyzed against phosphate-buffered saline (pH 7.0), and tested for bactericidal activity against selected target bacteria. Salmonella typhimurium LT-2 and a series of progressively rough lipopolysaccharide outer membrane mutants derived from it were used to monitor antimicrobial activity. Although an antimicrobial potential was present in rat granule contents for S. typhimurium, the growth of Pseudomonas aeruginosa PAO-1 in antimicrobial assay mixtures containing rat granule contents was substantially enhanced over control values. The growth enhancement property of the granule protein was heat resistant and promoted increased oxygen consumption by whole cells.     

The influence of cations on the permeability of the outer membrane of Salmonella typhimurium and other gram-negative bacteria.

The high sensitivity of rough mutants of Salmonella typhimurium, S. minnesota, and Escherichia coli 08 (i.e. with defects in the carbohydrate core of the lipopolysaccharide) to several antibiotics and to the dye gentian violet could be substantially reduced by the addition of cations (Mg2+, Na+) into the growth medium. One heptoseless mutant of S. typhimurium (chemotype Re) and its isogenic smooth parent strain were studied in more detail. The uptake of gentian violet was about 20% in the smooth strain, about 60% in the Re strain grown without additional cations, but decreased to about 15% in the same strain, when cations had been present during growth. In all cases, almost 50% of the gentian violet taken up by the cells was membrane-bound. The total membranes of the Re strain grown in nutrient broth without additional Mg2+ ions were reduced in the 36K and 34K major outer membrane proteins compared with the smooth strain; when grown with added cations the Re total membranes (and even whole cells) did not revert to the protein pattern of the smooth strain.     

Effect of high hydrostatic pressure on four genotypes of F-specific RNA bacteriophages

AIMS: The pressure responses of four genotypes of F-specific RNA bacteriophages, f2, GA, Qbeta and SP, were evaluated with respect to pressure magnitude, treatment temperature and suspending medium. METHOD AND RESULTS: The pressure responses were studied with respect to pressure magnitude (350 to 600 MPa), treatment temperature (-10 to 50 degrees C) and suspending media. Phages f2 and GA had much higher pressure resistances than Qbeta and SP. Pressure resistances of Qbeta and SP were enhanced with increase in salt concentrations in the range of 350 to 600 MPa from -10 to 50 degrees C in PBS. Qbeta and SP had greater pressure resistances when suspended in phosphate-buffered saline (PBS) with added glucose (5%, w/w), UHT whole milk and Dulbecco's Modified Eagle's Medium plus 10% fetal bovine sera than they did in PBS. Two surfactants, sucrose laurate and monolaurin, and one chelating agent, ethylenediamine tetraacetic acid (EDTA), increased the pressure resistance of Qbeta and SP, but had modest effect on either f2 or GA. CONCLUSIONS: Four representative F-specific RNA bacteriophages, f2 (serotype I), GA (serotype II), Qbeta (serotype III) and SP (serotype IV) showed different resistances to hydrostatic pressure in the range of 350-600 MPa. Significance and Impact of the Study: This study screened for practical surrogates of HAV for validation of commercial high hydrostatic pressure processing.     

Differential requirement of ATP and extra-ribosomal proteins for ribosome inactivation by eight RNA N-glycosidases.

The requirement of ATP and extra-ribosomal proteins for the inactivation of ribosomes by eight plant RNA N-glycosidases [ribosome-inactivating proteins (RIPs)] was investigated. Tritin, pokeweed antiviral protein and barley RIP depend, as gelonin [Sperti, S., Brigotti, M., Zamboni, M., Carnicelli, D. and Montanaro, L. (1991) Biochem. J., 277, 281-284], on the presence of ATP and extra-ribosomal proteins for full inactivation of ribosomes, while bryodin, lychnin, momordin, momorcochin and saporin inactivate isolated Artemia salina ribosomes suspended in buffer saline.