In vitro assay of Staphylococcus aureus enterotoxin A activity in food.

Staphylococcus aureus enterotoxin A (SEA) is a leading cause of food poisoning. The current test for functional activity of SEA requires monkeys or kittens. The major drawbacks of animal assays are lack of quantitation, poor reproducibility, low sensitivity, and high cost. In this report we describe and evaluate an alternative assay using T-cell proliferation to measure SEA activity in food. Human and rat lymphocytes proliferate in response to concentrations of SEA as low as 1 pg/ml, well below the pathogenic dose of 100 ng. This proliferation assay is highly sensitive, quantitative, and simple. Nonradioactive assays of T-cell proliferation were also suitable for detecting and measuring SEA, although with a 10-fold lower sensitivity. To evaluate the utility of this assay for food testing, four different food samples were mixed with SEA. In each sample, SEA was detected at a concentration of 1 ng/ml. Heat-inactivated SEA produced no detectable proliferation. These results demonstrate that an in vitro cell proliferation assay is an advantageous alternative to existing animal assays for measuring SEA activity in food.     

Microbial colonization of an in vitro model of a tissue engineered human skin equivalent – a novel approach

This was a preliminary investigation to define the conditions of colonization of a human skin equivalent (SE) model with cutaneous microorganisms. SEs of 24 mm diameter were constructed with a dermal matrix of fibrin containing fibroblasts and a stratified epidermis. Microbial colonization of the SEs was carried out in a dry environment, comparable to ' in vivo ' skin, using a blotting technique to remove inoculation fluid. The microbial communities were sampled by scrub washing and viable cells enumerated on selective growth medium. Staphylococcus epidermidis , Propionibacterium acnes and Malassezia furfur (human skin commensals) and Staphylococcus aureus (transient pathogen) were colonized at inoculum densities of 10²–10⁶ CFU SE⁻¹ on the surface of replicate SEs. Growth of all species was supported for upto 72–120 h, with recovery densities of between 10⁴–10⁹ CFU SE⁻¹. A novel, real-time growth monitoring method was also developed, using S. aureus containing a lux cassette. Light output increased from 20 to 95 h, and colonization increased from 10² to 10⁸ CFU SE⁻¹, as confirmed by conventional recovery. Thus, the SE model has potential to investigate interactions between resident and transient microbial communities with themselves and their habitat, and for testing treatments to control pathogen colonization of human skin.     

In Human Fetal Astrocytes Exposure to Interleukin-1β Stimulates Acquisition of the GD3+ Phenotype and Inhibits Cell Division

Abstract: In human astrocyte cultures established from second-trimester fetal brain tissue, ∼5–10% of total astrocyte population in unstimulated cultures were GD3⁺/glial fibrillary acidic protein (GFAP)⁺. The GD3⁺ cells were always GFAP⁺ and grew as flat, highly spread cells but changed to process-bearing cells after interleukin-1β (IL-1β) stimulation. It is interesting that IL-1β, a known mitogen for rat astrocytes, suppressed human fetal astrocyte proliferation as determined by [³H]thymidine incorporation, bromodeoxyuridine (BrdU) labeling, and cell counting. The GD3⁺ population, however, consistently increased in absolute number after IL-1β stimulation, in a dose- and time-dependent manner. The IL-1β-mediated increase in number of GD3⁺ astrocytes was independent of initial cell density or serum concentration. By flow cytometry, IL-1β enhanced both the mean fluorescence intensity and the percentage of GD3⁺ cells. To investigate whether the increase in GD3⁺ astrocyte cell number was due to proliferation of preexisting GD3⁺ astrocytes or due to conversion of GD3⁻ to GD3⁺ cells, we performed BrdU/GD3 double immunocytochemistry. BrdU/GD3 double-positive cells were extremely rare in both control and IL-1β-stimulated cultures. Moreover, an increase in number of GD3⁺ astrocytes was still observed in control and IL-1β-stimulated cultures where GD3⁺ cells had been initially eliminated by cell sorting. These results indicate that GD3⁺ astrocytes in human fetal culture may represent a postmitotic, differentiated, distinct phenotype.     

Influence of irradiance on in vitro growth and proliferation of Vaccinium corymbosum (highbush blueberry) and subsequent rooting in vivo

The effects of light on in vitro proliferation and subsequent in vivo rooting and acclimatisation of Vaccinium corymbosum were investigated. The shoots were exposed in vitro to different irradiances (total radiation ranging from 55 to 240 μmol m⁻² s⁻¹) for 7 to 60 days. In vitro growth and proliferation and the possible consequences on in vivo rooting were observed.
As compared to the control treatment (55 μmol m⁻² s⁻¹), higher irradiances improved proliferation and rooting ratios only with short applications (7 days). Short but high (210 μmol m⁻² s⁻¹) exposures applied at the end of the proliferation phase increased in vivo growth and rooting of the shoots. The shoots treated with strong light for longer times (14 and 28 days) showed both inhibition of growth and red colour of leaves and sprouts, and were less vigorous when transferred in vivo.     

Soybean root and nodule nitrate reductase

Nitrate reductase (NR) activity was followed in root and nodule from Glycine max (L.) Merr. (Cv. Tracy) inoculated with Rhizobium japonicum . Initially, a plus NO^3- in vivo assay was used. When chlorate-resistant mutants were used as inoculum, nodule NR activity was reduced by about 90%. indicating that the bacteroid accounts for much of the normal nodule's NR. With plants 3 to 15 weeks of age nodule NR activity (g fresh weight)^-1 was highest in young plants and root activity highest in old plants. Root and nodule total NR activity increased with plant age and were often not greatly different. Root NR activity correlated with plant NO^3- supply and increased from 0.8 to 11.4 μmol plant^-1 h^-1 as NO^3- was increased from 0 to 3 m M . In contrast, nodule NR activity was high in plants grown without NO^3- and did not appear to increase as nitrate supply to the plant was increased. Nodule activity was 6 to 14 μmol NO^2- plant^-1 h^-1. Use of a minus NO^3- in vivo assay had little affect on root NR activity, but greatly reduced nodule activity. Root tissue was found to have 5 to 38 times more NO^3- than nodule tissue. It is concluded that low nitrate levels within the nodule limit NR activity and that it is improbable that the nodule is a major site of plant nitrate reduction.     

Interaction between superantigen and T-cell receptor Vβ element determines levels of superantigen-dependent cell-mediated cytotoxicity of CD8+ T cells in induction and effector phases

Specific superantigens activate different T-cell fractions with distinct TCR Vβ elements in association with MHC class II molecules and also induce SDCC against MHC class II⁺ target cells. In the present study, to determine whether the responsiveness of each CD8⁺ T-cell fraction expressing a different TCR Vβ element is primarily determined by the TCR Vβ, we compared the levels of proliferation and SDCC in Vβ3⁺ and Vβ11⁺ T cells upon stimulation with SEA. Upon stimulation with SEA_wt, the levels of proliferation were higher in Vβ3⁺ T cells than in Vβ11⁺ T cells. The levels of SDCC were also higher for the combination of Vβ3⁺ T cells and SEA_wt than for the combination of Vβ11⁺ T cells and SEA_wt during both the induction phase and the effector phase. In addition, upon stimulation with SEA_m, the levels of proliferation were higher in Vβ11⁺ T cells than in Vβ3⁺ T cells. And then, the levels of SDCC were also higher for the combination of Vβ11⁺ T cells and SEA_m than for the combination of Vβ3⁺ T cells and SEA_m during both the induction phase and the effector phase. These results suggest that the SAG-responsiveness of each CD8⁺ T-cell fraction expressing a different TCR Vβ element is primarily determined by the interaction between the TCR Vβ element and the SAG.     

Induction of nitrate reductase in needles of Scots pine seedlings by NOX and NO3−

The possibility to induce nitrate reductase (NR; EC 1.6.6.2) in needles of Scots pine ( Pinus sylvestris L.) seedlings was studied. The NR activity was measured by an in vivo assay. Although increased NR activities were found in the roots after application of NO₃⁻, no such increase could be detected in the needles. Detached seedlings placed in NO₃⁻ solution showed increasing NR activities with increasing NO₃⁻ concentrations. Exposure of seedlings to NO_x (70–80 ppb NO₂ and 8–12ppb NO) resulted in an increase of the NR activity from 10–20 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹ to about 400 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. This level was reached after 2–4 days of exposure, thereafter the NR activity decreased to about 200 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. Analyses of free amino acids showed low concentrations of arginine and glutamine in NO_x-fumigated seedlings compared to corresponding controls.     

Opioid Modulation of Extracellular Signal-Regulated Protein Kinase Activity Is Ras-Dependent and Involves Gβγ Subunits

Abstract: Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with μ-, δ-, or κ-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding μ- ([ d -Ala²,Me-Phe⁴,Gly-ol⁵]enkephalin)-, δ- ([ d -Pen², d -Pen⁵]enkephalin)-, or κ- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a βγ scavenger, CD8-β-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8-β-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and βγ subunits of G_i/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.     

Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of σ32in vivo

The heat shock response of Escherichia coli is regulated by the cellular level and the activity of σ³², an alternative sigma factor for heat shock promoters. FtsH, a membrane-bound AAA-type metalloprotease, degrades σ³² and has a central role in the control of the σ³² level. The ftsH null mutant was isolated, and establishment of the Δ ftsH mutant allowed us to investigate control mechanisms of the stability and the activity of σ³² separately in vivo . Loss of the FtsH function caused marked stabilization and consequent accumulation of σ³² (≈20-fold of the wild type), leading to the impaired downregulation of the level of σ³². Surprisingly, however, Δ ftsH cells express heat shock proteins only two- to threefold higher than wild-type cells, and they also show almost normal heat shock response upon temperature upshift. These results indicate the presence of a control mechanism that downregulates the activity of σ³² when it is accumulated. Overproduction of DnaK/J reduces the activity of σ³² in Δ ftsH cells without any detectable changes in the level of σ³², indicating that the DnaK chaperone system is responsible for the activity control of σ³² in vivo . In addition, CbpA, an analogue of DnaJ, was demonstrated to have overlapping functions with DnaJ in both the activity and the stability control of σ³².     

COMPARISON OF LUMINOL CHEMILUMINESCENCE WITH ATP BIOLUMINESCENCE FOR THE ESTIMATION OF TOTAL BACTERIAL LOAD IN PURE CULTURES

A rapid chemiluminescent assay of total bacterial load that is based on the oxidation of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) as catalyzed by bacterial iron protoporphyrins is described and compared to the ATP bioluminescent assay of microbial biomass. An assay format that elicits linear light output response to a range of analyte concentrations of model compounds such as hematin and various heme-containing enzymes within the dynamic range of a BioOrbit 1251 luminometer is presented. When the assay was applied to eight pure bacterial cultures, the sensitivity was typically in the range of 10⁴-10⁵ cfu/ml, and was comparable to that obtained by the ATP assay. Similar levels of sensitivity can be derived from estimates of average values of 2.8 × 10^-18 mole of heme/cfu and 1 × 10^-19 mole of ATP/cfu. The potential of the luminal assay as an alternative rapid test for the estimation of total bacterial count in food and environmental samples is discussed.     

Nitrate reductase activity in nodules of pea inoculated with hydrogenase positive and hydrogenase negative strains of Rhizobium leguminosarum

The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup⁺) and hydrogenase negative (Hup⁻) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N₂-dependent or grown in the presence of 2 m M KNO₃. The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup⁻ strains accumulated more nitrite in the incubation medium than did those with Hup⁻ phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO₃ and inoculated with single Hup⁺ and Hup⁻ R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup⁻ strains in the R. leguminosarum -pea symbiosis.     

Effects of Tetanus Toxin on Catecholamine Release from Intact and Digitonin-Permeabilized Chromaffin Cells

Abstract: Tetanus exotoxin inhibited Ca²⁺-dependent cate-cholamine secretion in a dose-dependent manner in digito-nin-permeabilized chromaffin cells. The inhibition was specific for tetanus exotoxin and the B fragment of tetanus toxin; the C fragment had no effect. Inhibition required the introduction of toxin into the cell, and was not seen when intact cells were preincubated with the toxin or toxin fragments. The degree of inhibition was related to the length of preincubation with toxin, as well as the concentration of toxin used. A short preincubation with toxin was sufficient to inhibit secretion, and the continued presence of toxin in the incubation medium was not required during the incubation with Ca²⁺. The inhibition of secretion by tetanus toxin or the B fragment was not overcome with increasing Ca²⁺ concentrations. Tetanus toxin also inhibited catechol-amine secretion enhanced by phorbol ester-induced activation of protein kinase C. Thus, the toxin or a proteolytic fragment of the toxin can enter digitonin-permeabilized cells to interact with a component of the Ca²⁺-dependent exocytotic pathway to inhibit secretion.     

A method for measuring oxygen consumption in isolated perfused gills

A method is described for measuring respiration in isolated perfused flounder gills experiencing pressures and flows similar to those seen in vivo . Mean oxygen consumption of 13 preparations bathed and perfused in identical saline was 5·00 ± 0·75 (s.e.) μ mol h⁻¹ g wet⁻¹, whilst that of five preparations perfused with saline but bathed in sea water (32 mg l⁻¹) was 12·06±2·39 (s.e.) μmol h⁻¹ g wet⁻¹. The oxygen consumption of the seawater bathed gills was significantly higher (P<0·05) than that in saline bathed gills. These results provide direct evidence both of the high metabolic activity of the gill under normal perfusion conditions and of the increased energy expenditure of the giil in hyperosmotic, compared to isosmotic, environments.     

Physical characterization of the pore forming cytolysine from Gardnerella vaginalis

Abstract The cytolytic toxin (CTox) produced by Gardnerella vaginalis is able to form voltage-dependent cationic channels when incorporated in lipid membranes (Moran et al, 1991) FEBS Lett. 283, 317–320). Osmotic protection experiments show that toxin incorporated in human erythrocytes forms pores between 18 Å and 28 Å in diameter. A hypothesis of pore formation as a primary event to produce cytolysis is proposed. The CTox activity increases when cells are depolarized by increasing the extracellular K⁺ concentration, probably reflecting the voltage dependent character of CTox formed channels. The cytolytic effect of the toxin was prevented by low temperatures and was a function of the extracellular Ca²⁺ concentration, suggesting a Ca²⁺ influx as part of the lytic mechanism. Binding of CTox to erythrocytes was dependent on external Ca²⁺ and was less temperature-dependent. Dose-response analysis suggests cooperativity of the toxin for the lytic activity, although no direct evidence of oligomerization has been found.     

Simultaneous Measurement of Nitrite and Nitrate Levels as Indices of Nitric Oxide Release in the Cerebellum of Conscious Rats

Abstract: We examined the modulation of nitric oxide production in vivo by measuring levels of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the dialysate of the cerebellum in conscious rats, by using an in vivo brain microdialysis technique. The levels of both NO₂⁻ and NO₃⁻ were decreased by the intraperitoneal injection of N ^G-nitro- l -arginine methyl ester, an inhibitor of nitric oxide synthase, whereas N ^G-nitro- d -arginine methyl ester had no effect. l -Arginine by itself increased NO₂⁻ and NO₃⁻ levels and diminished the reduction of their levels caused by N ^G-nitro- l -arginine methyl ester. Direct infusion of l -glutamate, N -methyl- d -aspartate, or KCl into the cerebellum through a dialysis probe resulted in an increase in NO₂⁻ and/or NO₃⁻ levels. The effects of N -methyl- d -aspartate and KCl were dependent on extracellular calcium. Furthermore, the stimulatory effects of l -glutamate and N -methyl- d -aspartate were inhibited by N ^G-nitro- l -arginine methyl ester and (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an N -methyl- d -aspartate receptor antagonist. These results suggest that NO₂⁻ and NO₃⁻ levels may be related to nitric oxide production in vivo.     

Rapid and definitive detection of Salmonella in foods by PCR

The BAX^™ system for screening Salmonella is one of the first commercial PCR-based systems for the detection of food-borne pathogens. It is able to give a confirmed result within 28 h. There was 98·6% and 95·8% agreement between the BAX^™ system and conventional cultural analysis for the detection of Salmonella in artificially inoculated and uninoculated food samples, respectively. In both cases, the BAX^™ system generated more positive detections than cultural analysis. The speed of assay, ease of use and high specificity and sensitivity of the BAX^™ system for the detection of food-borne Salmonella make it an attractive method for routine food microbiology laboratories.     

Tetanus Toxin Inhibition of K+ -Stimulated [3H]GABA Release from Developing Cell Cultures of the Rat Cerebellum

Abstract: The effect of tetanus toxin pretreatment on K⁺ -stimulated [³H]γ-aminobutyric acid release from neuron-enriched cerebellar cell cultures at various stages during their development in vitro was assessed. Tetanus toxin had little inhibitory effect on immature (1-3-day-old) cultures, but markedly reduced K⁺-evoked [³H]γ-aminobutyric acid release from 7- and 14-day-old cultures (∼80% inhibition). It is suggested that cerebellar neurons in culture develop tetanus toxin-sensitive transmitter release mechanisms similar to their in vivo counterparts.     

A Comparison of Toxin Production by Clostridium botulinum Type E in Irradiated and Unirradiated Vacuum Packed Fish

S ummary . The rates of toxin production at 10° by inocula of 10⁵, 10⁴, 10³ and 10² spores of Clostridium botulinum type E in vacuum packed herring, cod and haddock were compared with that of equivalent numbers of spores surviving γ-irradiation at 0.3 Mrad. There was little difference between the rates of toxin production in unirradiated and irradiated fish. More toxin was produced in irradiated herring than in unirradiated, but in haddock the reverse was obtained. In cod about equal amounts of toxin were found without trypsinization; after trypsinization toxin levels were generally higher in irradiated samples.     

GTP-specific pyrophosphorylation of thiamin in dark-grown soybean (Glycine max) seedling axes

ATP:thiamin pyrophosphotransferase (TPT: EC 2.7.6.2) was purified 5 900-fold from 48 h dark-grown soybean [ Glycine max (L.), Merr. cv. Ransom II] seedling axes. TPT activity was monitored during purification by measuring the formation of thiamin pyrophosphate (TPP) from [2-¹⁴C]-thiamin at optimal pH (7.3). Although other nucleoside triophosphates were active as pyrophosphate donors (apparent K_ms from 21 to 138 m M ), GTP was the preferred nucleotide with an apparent K_m of 0.021 m M . TPT activity was extremely sensitive to TPP formation, suggesting product feedback inhibition of TPT activity in vivo. Sulfhydryl, H⁺ and Mg²⁺ concentrations, either independently or in concert, were found to affect TPT activity.     

Denitrification and nitrate-reducing bacterial populations in abandoned and reclaimed minesoils

Abstract Microbial populations, nitrogen mineralization potentials, and denitrification enzyme activities were examined in two abandoned carbolithic minesoils. Numbers and activities of bacteria and fungi were lower in nonamended than in lime and/or fly ash amended sites. Rates of aerobic NO₃⁻ production (3 to 38 μg-N kg⁻¹ h⁻¹) and anaerobic NO₃⁻ reduction to N₂O (5 to 68 μg-N kg⁻¹ h⁻¹) were measured. Organisms capable of N₂O production under anaerobic soil conditions were present in low numbers, and their activity was restricted in part by low soil pH. Nondenitrifying nitrate-reducing bacteria were more diverse and in greater numbers than respiratory denitrifiers and may have been responsible for N₂O production in assays measuring denitrification enzyme activity.