Growth and Multiplexed Analysis of Microorganisms on a Subdivided, Highly Porous, Inorganic Chip Manufactured from Anopore

A highly porous inorganic material (Anopore) was shown to be an effective support for culturing and imaging a wide range of microorganisms. An inert barrier grid was printed on the rigid surface of Anopore to create a “living chip” of 336 miniaturized compartments (200/cm²) with broad applications in microbial culture.     

Use of Inorganic Membrane Filters (Anopore) for Epifluorescence and Scanning Electron Microscopy of Nanoplankton and Picoplankton

Inorganic membrane filters (Anopore) were examined qualitatively by epifluorescence and scanning electron microscopy to determine their suitability for the study of nanoplankton and picoplankton. Compared with filters currently used, the Anopore filters allowed for increased resolution of the specimen with epifluorescence microscopy because of filter flatness and increased illumination caused by the large number of pores cm^-2. The inorganic filters had a lower filtration rate than polycarbonate filters. For scanning electron microscopy, the metal oxide (Anopore) filters were efficient support for the plankton, with little charging of cells or background.     

Ability of various inserts to promote endothelium cell culture for the establishment of coculture models

To select an insert suitable for human umbilical vein endothelial cell (HUVEC) culture, we compared several available inserts of 0.2 to 0.45 m porosity: Cellagen (ICN), Transwell-COL (Costar), Millicell-HA and CM (Millipore), Anopore (Nunc), Cyclopore (Falcon) in comparison with a control surface (Thermanox). The requirements were: (i) to promote attachment, adhesion and proliferation of HUVEC (judged by [³H]thymidine incorporation into DNA at days 1, 3, 7); (ii) to allow HUVEC visualization by inverted, fluorescence microscopy for uptake of DiI-Ac-LDL and scanning electron microscopy, performed at day 9 after seeding.Because Transwell and Cellagen are collagen precoated and CM has to be coated for cell culture, we performed collagen coating (types I + III or IV) for non-pretreated inserts for the purpose of comparison. Our preferences comprise Transwell-COL, Cyclopore not coated or coated (whatever the collagen type), and Cellagen. However, on a quality/price ratio criterion, Cyclopore, even uncoated, is the insert of choice. The HA, CM and Anopore inserts, even coated, do not allow HUVEC growth but do not alter positive uptake of acetylated LDL.Abbreviations HUVEC human umbilical vein endothelial cells     

Immobilization of alliinase on porous aluminum oxide

Membrane filters prepared from porous aluminum oxide (Anopore) were investigated for their potential use as a durable support for enzymes. Alliinase (EC 4.4.1.4) was chosen as a model enzyme for immobilization experiments. To allow for smooth fixation, the enzyme was immobilized indirectly by sugar-lectin binding. Monomolecular layers of the lectin concanavalin A and alliinase were applied by self-assembling processes. As an anchor for these layers, the sugar, mannan, was covalently coupled to the membrane surface. This procedure exhibits several advantages: (i) enzyme immobilization can be carried out under smooth conditions; (ii) immobilization needs little time; and (iii) protein layers may be renewed.     

Quantitative analysis of population heterogeneity of the adaptive salt stress response and growth capacity of Bacillus cereus ATCC 14579

Bacterial populations can display heterogeneity with respect to both the adaptive stress response and growth capacity of individual cells. The growth dynamics of Bacillus cereus ATCC 14579 during mild and severe salt stress exposure were investigated for the population as a whole in liquid culture. To quantitatively assess the population heterogeneity of the stress response and growth capacity at a single-cell level, a direct imaging method was applied to monitor cells from the initial inoculum to the microcolony stage. Highly porous Anopore strips were used as a support for the culturing and imaging of microcolonies at different time points. The growth kinetics of cells grown in liquid culture were comparable to those of microcolonies grown upon Anopore strips, even in the presence of mild and severe salt stress. Exposure to mild salt stress resulted in growth that was characterized by a remarkably low variability of microcolony sizes, and the distributions of the log(10)-transformed microcolony areas could be fitted by the normal distribution. Under severe salt stress conditions, the microcolony sizes were highly heterogeneous, and this was apparently caused by the presence of both a nongrowing and growing population. After discriminating these two subpopulations, it was shown that the variability of microcolony sizes of the growing population was comparable to that of non-salt-stressed and mildly salt-stressed populations. Quantification of population heterogeneity during stress exposure may contribute to an optimized application of preservation factors for controlling growth of spoilage and pathogenic bacteria to ensure the quality and safety of minimally processed foods.     

Quantitative Analysis of Population Heterogeneity of the Adaptive Salt Stress Response and Growth Capacity of Bacillus cereus ATCC 14579

Bacterial populations can display heterogeneity with respect to both the adaptive stress response and growth capacity of individual cells. The growth dynamics of Bacillus cereus ATCC 14579 during mild and severe salt stress exposure were investigated for the population as a whole in liquid culture. To quantitatively assess the population heterogeneity of the stress response and growth capacity at a single-cell level, a direct imaging method was applied to monitor cells from the initial inoculum to the microcolony stage. Highly porous Anopore strips were used as a support for the culturing and imaging of microcolonies at different time points. The growth kinetics of cells grown in liquid culture were comparable to those of microcolonies grown upon Anopore strips, even in the presence of mild and severe salt stress. Exposure to mild salt stress resulted in growth that was characterized by a remarkably low variability of microcolony sizes, and the distributions of the log₁₀-transformed microcolony areas could be fitted by the normal distribution. Under severe salt stress conditions, the microcolony sizes were highly heterogeneous, and this was apparently caused by the presence of both a nongrowing and growing population. After discriminating these two subpopulations, it was shown that the variability of microcolony sizes of the growing population was comparable to that of non-salt-stressed and mildly salt-stressed populations. Quantification of population heterogeneity during stress exposure may contribute to an optimized application of preservation factors for controlling growth of spoilage and pathogenic bacteria to ensure the quality and safety of minimally processed foods.     

Comparison of a new inorganic membrane filter (Anopore) with a track-etched polycarbonate membrane filter (Nuclepore) for direct counting of bacteria.

Bacterial counts obtained by using a new Anopore inorganic membrane filter were 21 to 33% higher than those obtained by using a Nuclepore polycarbonate membrane filter. In addition, the inorganic filter had higher flow rates, permitting lower vacuum pressures to be used, while the intrinsically flat, rigid surface resulted in easier focusing and sharp definition of bacteria across the whole field of view.     

Comparison of a new inorganic membrane filter (Anopore) with a track-etched polycarbonate membrane filter (Nuclepore) for direct counting of bacteria

Bacterial counts obtained by using a new Anopore inorganic membrane filter were 21 to 33% higher than those obtained by using a Nuclepore polycarbonate membrane filter. In addition, the inorganic filter had higher flow rates, permitting lower vacuum pressures to be used, while the intrinsically flat, rigid surface resulted in easier focusing and sharp definition of bacteria across the whole field of view.     

Properties of microfiltration membranes: the effects of adsorption and shear on the recovery of an enzyme

An experimental study of the interaction of the enzyme yeast alcohol dehydrogenase (YADH) with microfiltration membranes has been carried out. Most measurements were made with capillary pore inorganic membranes (Anopore) with some comparative measurements being made with polymeric membranes of low protein affinity (Durapore). It has been shown that the prolonged exposure of the enzyme to the inorganic membrane under low-shear conditions (slow recycle) resulted in a loss of enzyme activity. Under filtration conditions, the membrane permeation rate decreased continuously with time. This decrease could be quantified using the standard blocking filtration law, which describes a decrease in pore volume due to deposition of enzyme on the walls of the pore. No significant loss in activity of permeating enzyme occurred under solution conditions where the enzyme was stable. However, a significant loss of such activity occurred under solution conditions where the enzyme was slightly unstable. The experiments indicate that the likely mechanism for activity loss is a membrane/enzyme interaction resulting from a shear induced deformation of the enzyme structure. Two conclusions of practical importance are drawn from the work. (c) 1992 John Wiley & Sons, Inc.     

Prolonged in vitro cultivation of Ichthyophthirius multifiliis using an EPC cell line as substrate

The ciliate Ichthyophthirius multifiliis, which normally requires a fish host to develop from the theront stage to the trophont stage, was cultivated in vitro for part of its life cycle. Experiments were conducted using a laboratory strain of the parasite originally isolated from rainbow trout Oncorhynchus mykiss in a Danish trout farm. Theronts escaping from tomontocysts were kept in water, cell culture media (E-MEM or L-15), or cultures of EPC (Epithelioma Papulosum Cyprini) cells in plastic tissue culture dishes (Nunc multidish plates). In addition, a 2-compartment system, with water separated from tissue culture media by a monolayer of EPC cells on an Anopore Tissue Culture Insert (mimicking the fish epidermis) was tested as an experimental habitat for the parasite. Theronts transformed into trophonts in all treatments except in water alone. However, development was accelerated in wells containing EPC cells, and survival and growth of trophonts were significantly increased compared to water or tissue culture media alone. Further, the 2-compartment system allowed superior performance of the parasites (attachment of parasites to cells and growth from 36 to 46 microm). In all experiments it was found that the presence of host factors (mucus and serum) stimulated parasite development.     

Direct-Imaging-Based Quantification of Bacillus cereus ATCC 14579 Population Heterogeneity at a Low Incubation Temperature

Bacillus cereus ATCC 14579 was cultured in microcolonies on Anopore strips near its minimum growth temperature to directly image and quantify its population heterogeneity at an abusive refrigeration temperature. Eleven percent of the microcolonies failed to grow during low-temperature incubation, and this cold-induced population heterogeneity could be partly attributed to the loss of membrane integrity of individual cells.Bacillus cereus is a food poisoning- and food spoilage-causing organism that can be found in a large variety of foods (4, 23). There are two illnesses associated with B. cereus, namely, emetic and diarrheal intoxication (17, 24). Most of the strains related to cases or outbreaks of B. cereus food-borne poisoning were shown to be unable to grow at 7°C (1, 12). The average temperatures of domestic refrigerators have been investigated in various surveys around the world and often ranged from 5°C to 7°C, but extreme values exceeded 10°C to 12°C (5, 16). Inadequate chilling was indeed reported in various incidents of B. cereus food-borne illness (7, 8, 18, 19), pointing to the importance of appropriate refrigeration of foods contaminated with B. cereus to control its growth and toxin production in foods (9).Several studies have demonstrated that microorganisms can show diversity in their population stress response, even in an apparently homogeneous stress environment (6, 11, 21, 22). However, only very limited data describing the heterogeneity in growth performance of individual cells from food-borne pathogens cultured at low temperatures are available (10). Because inadequate chilling of food is one of the factors that contribute to the number of incidents of B. cereus food-borne illness, there is a need for better understanding of its growth performance at lowered incubation temperatures. In this study, we used the direct-imaging-based Anopore technology (6, 13-15) to quantitatively describe the population heterogeneity of B. cereus ATCC 14579 cells at 12°C. The minimum temperature for the growth of B. cereus ATCC 14579 in brain heart infusion (BHI) broth is 7.5°C (personal communication from F. Carlin), but various food-borne-associated B. cereus isolates were shown to be unable to grow at 10°C (1). Therefore, in this study, a culturing temperature of 12°C was chosen, to mimic temperature abuse of refrigerated foods. In addition, the membrane integrity of individual cells was assessed using both membrane permeant and impermeant nucleic acid dyes in order to get more insight into cellular characteristics that may contribute to heterogeneity in growth response.     

Functional symbiosis between endothelium and epithelial cells in glomeruli

In some capillary beds, pericytes regulate endothelial growth. Capillaries with high filtration capacity, such as those in renal glomeruli, lack pericytes. Glomerular endothelium lies adjacent to visceral epithelial cells (podocytes) that are anchored to and cover the anti-luminal surface of the basement membrane. We have tested the hypothesis that podocytes can function as endothelial supporting cells. Endothelial cells were outgrown from circulating endothelial progenitors of normal subjects and were extensively characterized. These blood outgrowth endothelial cells (BOECs) expressed endothelial markers, lacked stem cell markers, and expressed the angiopoietin-1 receptor, Tie-2, and the vascular endothelial growth factor (VEGF) receptor, Flk-1. Differentiated podocytes in culture expressed and secreted VEGF, which was upregulated 4.5-fold by high glucose. In complete medium, BOECs formed thin cell-cell connections and multicellular tubes on Matrigel, the in vitro correlate of angiogenesis. This was impaired in deficient media but rescued by co-incubation with Transwell Anopore inserts containing differentiated podocytes. To assess whether VEGF was the major podocyte-derived signal that rescued BOEC angiogenesis, we examined angiogenesis of control and Flk-1-deficient BOECs. Co-incubation with podocytes or addition of recombinant VEGF each rescued angiogenesis in control BOECs, but both failed to support maintenance and angiogenesis in Flk-1-deficient BOECs. Finally, co-culture with podocytes increased BOEC-proliferation. In concert, these findings suggest a model in which glomerular visceral epithelial cells function as pericyte-like endothelial supporting cells. Podocyte-derived VEGF is a required and sufficient regulator of vascular endothelial maintenance, and its upregulation in podocytes by high glucose may be the mechanism for the increased glomerular angiogenesis that is observed in vivo in early diabetic glomerular injury. These studies were supported by grants from the National Institutes of Health (NIH-NIDDK 63360) and the Juvenile Diabetes Research Foundation (JDRF-1-2004-78).     

Fouling of microfiltration membranes by broth-free antifoam agents

The fouling tendencies of seven commercial antifoam agents used with microfiltration membranes were investigated in a stirred cell. Parameters such as viscosity, oil droplet size distribution, contact angle, work of adhesion (W(a)), membrane type, operating pressure, and feed concentration were examined. The results show that a silicone-based antifoam, G832, gave a significantly lower flux (相似文献   

Modeling of basolateral ATP release induced by hypotonic treatment in A6 cells

ATP is released from the basolateral membrane of A6 epithelia in response to hypotonic treatment. This study addresses the problem of ATP diffusion through the permeable supports used to culture the cells. A theoretical analysis of a recently introduced experimental protocol is presented and a model of ATP diffusion through the compartments of the measuring system is proposed. The model provides the ATP profiles near the cell layer and in the measurement chamber. Comparison of results from computer simulations and experimental data showed that the permeable support introduces a marked delay for ATP diffusion, supporting the correlation of apparently time-separated events: the mobilization of Ca²⁺ from internal stores and release of ATP from the cell. The model is consistent with experimental data obtained with the luciferin–luciferase pulse protocol and provides an indirect proof of related processes like the closure and opening of the lateral interspace that occur after imposing the hyposmotic shock. The influence of the pore structure of the permeable support in modulating the measured release rates revealed by computer simulation is experimentally validated for two types of Anopore filters.     

Protein Localization in Escherichia coli Cells: Comparison of the Cytoplasmic Membrane Proteins ProP,LacY, ProW,AqpZ, MscS,and MscL

Fluorescence microscopy has revealed that the phospholipid cardiolipin (CL) and FlAsH-labeled transporters ProP and LacY are concentrated at the poles of Escherichia coli cells. The proportion of CL among E. coli phospholipids can be varied in vivo as it is decreased by cls mutations and it increases with the osmolality of the growth medium. In this report we compare the localization of CL, ProP, and LacY with that of other cytoplasmic membrane proteins. The proportion of cells in which FlAsH-labeled membrane proteins were concentrated at the cell poles was determined as a function of protein expression level and CL content. Each tagged protein was expressed from a pBAD24-derived plasmid; tagged ProP was also expressed from the chromosome. The osmosensory transporter ProP and the mechanosensitive channel MscS concentrated at the poles at frequencies correlated with the cellular CL content. The lactose transporter LacY was found at the poles at a high and CL-independent frequency. ProW (a component of the osmoregulatory transporter ProU), AqpZ (an aquaporin), and MscL (a mechanosensitive channel) were concentrated at the poles in a minority of cells, and this polar localization was CL independent. The frequency of polar localization was independent of induction (at arabinose concentrations up to 1 mM) for proteins encoded by pBAD24-derived plasmids. Complementation studies showed that ProW, AqpZ, MscS, and MscL remained functional after introduction of the FlAsH tag (CCPGCC). These data suggest that CL-dependent polar localization in E. coli cells is not a general characteristic of transporters, channels, or osmoregulatory proteins. Polar localization can be frequent and CL independent (as observed for LacY), frequent and CL dependent (as observed for ProP and MscS), or infrequent (as observed for AqpZ, ProW, and MscL).Modern developments in fluorescence microscopy have led to a new understanding of the organization of bacterial cells, particularly protein and lipid localization (21, 56). Analysis of the subcellular localization of diverse proteins and lipids has shown that they are not uniformly distributed. The phospholipid cardiolipin (CL) localizes at the poles and septal regions (36), and there is evidence for segregation of phosphatidylethanolamine (PE) from phosphatidylglycerol (PG) in the membranes of living Escherichia coli cells (69). Localization of many proteins that are integral or peripheral to the cytoplasmic membrane has been studied by fusing them to green fluorescent protein (GFP) (or its derivatives), and it is possible to classify the fusion proteins according to their subcellular localization. The first group, comprised of proteins that are concentrated at the cell poles, includes chemoreceptors (31, 62), the lactose permease LacY (43), and the metabolic sensor kinases DcuS and CitA (55). Members of the second group form helices that extend from pole to pole and include MreB (25), MinD (57), the Sec protein export system (58), and RNase E, which is the main component of the RNA degradosome in E. coli (67). Other proteins may appear to be similarly distributed due to their association with the Sec system (58). Members of the third group are uniformly distributed and include the mechanosensitive channel MscL (45) and the sensor kinase KdpD (32).The polar localization of proteins appears to be a critical feature of the complicated internal localization of bacteria. For example, it is important for temporally and spatially accurate placement of the septum during cell division (15). However, the mechanism of protein organization at bacterial cell poles is still unclear, and in many cases its functional role has not been determined. Do the poles merely serve as a receptacle for proteins, superstructures, or membrane domains with no functional effects, or is this location functionally important for membrane proteins and lipids?Recent evidence indicates that the subcellular localization of the transporter ProP in E. coli is related to membrane phospholipid composition, cardiolipin localization, and ProP function (51, 52). E. coli cells from cultures grown to exponential phase contain mostly the zwitterionic phospholipid PE (approximately 75 mol%) and the anionic phospholipids PG (approximately 20 mol%) and CL (approximately 5 mol%) (8). (Note that cardiolipin is diphosphatidylglycerol.) However, the phospholipid composition depends on the bacterial growth conditions. We found that the proportion of CL among E. coli lipids varies directly with growth medium osmolality (68), and increased CL synthesis was at least partially attributed to regulation of the cls locus encoding cardiolipin synthase (52). There is residual CL in cls bacteria, indicating that there is an alternative pathway for CL synthesis (51). The CL-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) was used to show that CL clusters at the poles and septa in growing E. coli cells (36, 52). This result was corroborated by analyzing the phospholipid composition of E. coli minicells (DNA-free cells resulting from asymmetric cell division) (24, 51).ProP is an osmosensory transporter that senses increasing osmolality and responds by mediating the cytoplasmic accumulation of organic osmolytes (e.g., proline, glycine betaine, and ectoine). Biochemical regulation of the ProP protein ensures that ProP activity increases with increasing assay medium osmolality (49). We showed that ProP and CL colocalize at the poles and near the septa of dividing E. coli cells and that the polar concentration of ProP correlates with the polar concentration of CL (52). Moreover, we showed that the osmolality required to activate ProP increased in parallel to the CL content when E. coli was cultivated in media with increasing osmolality (51, 52, 68). The osmolality required to activate ProP was also a direct function of CL content in proteoliposomes reconstituted with purified ProP (51). We concluded that concentration at the cell poles controlled the osmoregulatory function of ProP by placing the transporter in a cardiolipin-rich environment.To determine whether CL-dependent membrane protein localization is a general phenomenon in E. coli, we compared the subcellular localization of ProP with that of its paralogue LacY, a well-characterized lactose transporter (16). LacY and ProP are both members of the major facilitator superfamily and H⁺ symporters. LacY transports the nutrient lactose, and LacY activity decreases while ProP activity increases with increasing osmolality (9). Nagamori et al. reported polar localization of a LacY-GFP fusion protein in E. coli (43). We confirmed this observation and demonstrated that, in contrast to the behavior of ProP, the polar concentration of LacY did not correlate with the polar concentration of CL (51).In this work we further explored the relationship between CL and protein localization in E. coli. We compared ProP with other proteins related to cellular osmoregulation. Bacteria use arrays of osmoregulatory mechanisms to survive and function when the osmotic pressure of their environment changes. In E. coli, the aquaporin AqpZ mediates transmembrane water flux, the transporters ProP, ProU, BetT, and BetU mediate organic osmolyte accumulation at high osmotic pressure, and the mechanosensitive (MS) channels MscL and MscS mediate solute efflux in response to osmotic downshock (71). Localization of these proteins might be expected since AqpZ might influence cell morphology changes by accelerating water flux at particular positions on the cell surface and the pressure sensitivities of MscL and MscS are known to depend on membrane curvature in vitro (18).For ProP and LacY, we labeled the inserted peptide tag CCPGCC with the biarsenical fluorescein reagent FlAsH-EDT₂ (fluorescein arsenical helix binder, bis-EDT adduct) (1, 2) to examine the subcellular localization of AqpZ, the integral membrane component ProW of the osmoregulatory ATP-binding cassette (ABC) transporter ProU, and the MS channel proteins MscS and MscL in cls⁺ and cls bacteria. Fluorescence microscopy was used to determine the proportion of cells with labeled protein concentrated at the poles as a function of bacterial CL content and protein expression level. For ProP, the frequency with which MscS was concentrated at cell poles was proportional to the level and polar concentration of CL. LacY concentrated at the cell poles at a high and CL-independent frequency. The frequencies with which AqpZ, MscL, and ProW concentrated at the cell poles and septa were low (up to 12%) and CL independent.     

Carbon isotope discrimination,water-use efficiency,growth, and mortality in a natural shrub population

In order to scale up from the ecophysiological characters of individual plants to population-level questions, we need to determine if character patterns in natural populations are stable through time, and if the characters are related to growth and survival. We investigated these questions in a 3-year study for one character, integrated water-use efficiency (WUE) as estimated by carbon isotope discrimination () in a population of the Great Basin shrub, Chrysothamnus nauseosus. WUE was a conservative character for a given plant within and across seasons, and a previously documented difference between two size classes (represented by juveniles and adults) was maintained; smaller juveniles had a lower WUE than larger adults. The lower WUE of juveniles was often accompanied by higher rates of photosynthesis and stomatal conductance as compared to adults even though juveniles generally had more negative xylem pressure potentials. Although many discussions of the role of WUE in natural populations have been based on the expectation that higher WUE (lower ) is generally associated with less growth, we found no such relation-ship for juvenile plants in this population (i.e was not positively correlated with height increase). In addition, juvenile plant mortality was not correlated with . Although there were stable patterns of WUE for plants in this population, the positive correlation between WUE and size, and the lack of a negative correlation between WUE and height growth, make it unlikely that the WUE of an individual plant will be related in a simple manner to its growth and survival in the population.     

Identification of the bacteriochlorophylls, carotenoids, quinones, lipids, and hopanoids of "Candidatus Chloracidobacterium thermophilum"

"Candidatus Chloracidobacterium thermophilum" is a recently discovered chlorophototroph from the bacterial phylum Acidobacteria, which synthesizes bacteriochlorophyll (BChl) c and chlorosomes like members of the green sulfur bacteria (GSB) and the green filamentous anoxygenic phototrophs (FAPs). The pigments (BChl c homologs and carotenoids), quinones, lipids, and hopanoids of cells and chlorosomes of this new chlorophototroph were characterized in this study. "Ca. Chloracidobacterium thermophilum" methylates its antenna BChls at the C-8(2) and C-12(1) positions like GSB, but these BChls were esterified with a variety of isoprenoid and straight-chain alkyl alcohols as in FAPs. Unlike the chlorosomes of other green bacteria, "Ca. Chloracidobacterium thermophilum" chlorosomes contained two major xanthophyll carotenoids, echinenone and canthaxanthin. These carotenoids may confer enhanced protection against reactive oxygen species and could represent a specific adaptation to the highly oxic natural environment in which "Ca. Chloracidobacterium thermophilum" occurs. Dihydrogenated menaquinone-8 [menaquinone-8(H(2))], which probably acts as a quencher of energy transfer under oxic conditions, was an abundant component of both cells and chlorosomes of "Ca. Chloracidobacterium thermophilum." The betaine lipid diacylglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine, esterified with 13-methyl-tetradecanoic (isopentadecanoic) acid, was a prominent polar lipid in the membranes of both "Ca. Chloracidobacterium thermophilum" cells and chlorosomes. This lipid may represent a specific adaptive response to chronic phosphorus limitation in the mats. Finally, three hopanoids, diploptene, bacteriohopanetetrol, and bacteriohopanetetrol cyclitol ether, which may help to stabilize membranes during diel shifts in pH and other physicochemical conditions in the mats, were detected in the membranes of "Ca. Chloracidobacterium thermophilum."     

Effects of Source Water Quality on Chlorine Inactivation of Adenovirus,Coxsackievirus, Echovirus,and Murine Norovirus

More information is needed on the disinfection efficacy of chlorine for viruses in source water. In this study, chlorine disinfection efficacy was investigated for USEPA Contaminant Candidate List viruses coxsackievirus B5 (CVB5), echovirus 1 (E1), murine norovirus (MNV), and human adenovirus 2 (HAdV2) in one untreated groundwater source and two partially treated surface waters. Disinfection experiments using pH 7 and 8 source water were carried out in duplicate, using 0.2 and 1 mg/liter free chlorine at 5 and 15°C. The efficiency factor Hom (EFH) model was used to calculate disinfectant concentration × contact time (CT) values (mg·min/liter) required to achieve 2-, 3-, and 4-log₁₀ reductions in viral titers. In all water types, chlorine disinfection was most effective for MNV, with 3-log₁₀ CT values at 5°C ranging from ≤0.020 to 0.034. Chlorine disinfection was least effective for CVB5 in all water types, with 3-log₁₀ CT values at 5°C ranging from 2.3 to 7.9. Overall, disinfection proceeded faster at 15°C and pH 7 for all water types. Inactivation of the study viruses was significantly different between water types, but no single source water had consistently different inactivation rates than another. CT values for CVB5 in one type of source water exceeded the recommended CT values set forth by USEPA''s Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water Systems using Surface Water Sources. The results of this study demonstrate that water quality plays a substantial role in the inactivation of viruses and should be considered when developing chlorination plans.Disinfection processes are critical for the reduction of infectious virus concentrations in source water, because viruses are less efficiently removed by primary treatment of drinking water (e.g., coagulation and filtration) than are other pathogen types of concern (e.g., bacteria and protozoa). Over the years, many disinfection studies have focused on the inactivation of viruses in purified and buffered, demand-free, reagent-grade water (RGW). However, relatively few investigators have examined the impact of water quality during the disinfection process, even though water quality has been found to be a significant factor for inactivation of viruses.Several researchers found that the inactivation rate of poliovirus by free chlorine increased as the ionic concentration of water increased. In one study, poliovirus 1 was inactivated three times faster in boric acid buffer than in purified water (3). In addition, several investigators found that when the ionic content of buffered water was raised by the addition of NaCl or KCl, poliovirus 1 was inactivated two to four times faster than in the buffered water alone (2, 16, 17). In another study, poliovirus 1 was inactivated 10 times more rapidly in drinking water than in purified water (4).Studies conducted with natural waters have demonstrated both increased and decreased disinfection efficacy of chlorine in these waters compared to purified or buffered waters. In a study comparing chlorine disinfection in purified water and Potomac estuarine water, coxsackievirus A9 was inactivated more rapidly in the source water. The remaining study viruses (coxsackievirus B1, echovirus 7, adenovirus 3, poliovirus 1, and reovirus 3) were all inactivated more slowly in the source water (13). Bacteriophage MS2 was inactivated more slowly by free chlorine in two types of surface water than in buffered, demand-free water. However, there was no difference between the inactivation rates of this virus in the buffered water and groundwater (10). In another study, both feline calicivirus and adenovirus 40 were inactivated more slowly in treated groundwater than in buffered, demand-free water (21).The United States Environmental Protection Agency''s (USEPA) Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water Systems using Surface Water Sources (Guidance Manual) recommends disinfectant concentration × contact time (CT) values of 4, 6, and 8 to achieve 2-, 3-, and 4-log₁₀ inactivation, respectively, with chlorine at 5°C and pH 6 to 9 (23). These CT values, which incorporate a safety factor of 3, were obtained from inactivation experiments conducted with monodispersed hepatitis A virus (HAV) in buffered, demand-free water. As water quality can significantly affect the disinfection efficacy of chlorine, it is unclear whether these CT value recommendations are sufficient for inactivation of viruses in source water. More information is needed to systematically examine the role of water quality in chlorine disinfection of viruses.The objective of the present study was to examine the disinfection efficacy of free chlorine on selected viruses from USEPA''s Contaminant Candidate List (CCL) (22) in one untreated and two partially treated source waters from distinct geographical regions. By comparing the efficacy of chlorine disinfection in the source water types to disinfection in buffered, chlorine-demand-free RGW (7), the impact of water quality could be examined. The four representative CCL viruses selected for this study included human adenovirus 2 (HAdV2), echovirus 1 (E1), coxsackievirus B5 (CVB5), and murine norovirus (MNV), a surrogate for human norovirus (22). The viruses were selected because they were previously found to be the least effectively inactivated viruses of their type in RGW (6). Disinfection experiments were carried out in duplicate in pH 7 and 8 source water at 5 and 15°C using 0.2 and 1 mg/liter free chlorine. Inactivation curves were plotted using Microsoft Excel, and CT values were calculated using the efficiency factor Hom (EFH) model (9).     

Agonist function of the neurokinin receptor antagonist, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P,in monocytes

G-protein-coupled bombesin receptors are capable of signaling through the G(i) protein even when receptor-coupling to G(q) is blocked by [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P (SpD), a neurokinin-1 receptor antagonist and "biased" agonist to bombesin receptors. As bombesin is a monocyte and tumor cell attractant, we were interested in the effects of SpD on cell migration. Chemotaxis of monocytes was tested in micropore filter assays. SpD was a dose-dependent agonist in monocyte migration and was not inhibited by antagonists to neurokinin-1 or -2 receptors. SpD failed to inhibit chemotaxis toward bombesin, suggesting that inhibition of bombesin receptor coupling to G(q) with SpD does not impair migratory responses elicited by bombesin. As pertussis toxin inhibited migration, coupling of receptors to G(i) may signal migration. Chemotaxis toward SpD was inhibited by bombesin receptor antagonists as well as by blocking signaling enzymes downstream of G(q) (phospholipase-3 and protein kinase C with wortmannin and bisindolylmaleimide, respectively), suggesting transactivation of G(q)-mediated chemotaxis signaling by SpD via bombesin receptors. Protein kinase C that induces sphingosine kinase activation and production of sphingosine-1-phosphate, which may lead to G(q)-dependent chemoattraction, was involved in SpD-dependent migration. Inhibition of sphingosine-1-phosphate production with dimethylsphingosine inhibited monocyte migration toward SpD. Data suggest that SpD induces migration in monocytes and signaling events involving activation of sphingosine kinase in a G(i) protein- and protein kinase C-dependent fashion. "Biased" agonism of SpD at bombesin receptors may affect normal and tumor cell migration.     

Molecular weights,poly(A)-content,and partial separation of chick globin mRNAs

Poly(A)-containing 9S RNA from chick reticulocytes was electrophoresed on formamide-polyacrylamide gels. The molecular weight was determined to be 211 000±10 000 daltons. The RNA was separated into three different fractions with respect to molecular weight. These RNAs were translated in a wheat germ cell-free system. The lower molecular weight RNA directed up to 95% -chain synthesis, compared to 60% for the higher molecular weight RNA. This was accompanied by a relative increase for -chain synthesis with increasing molecular weight. It could also be shown by hybridization with labelled poly(U) that the average poly(A) length decreased from about 83 nucleotides for fraction I to 36 nucleotides for fraction III. Our results suggest that fractionation of avian 9 S globin mRNA by electrophoresis on formamide-polyacrylamide gels is dependent upon two parameters, namely differences in the lengths of the non-poly(A)-containing portion of the and mRNAs and differences in the poly(A) lengths.