Measurement of virulence of aeromonads using a suckling mouse model of infection

Abstract Virulent and avirulent strains of Aeromonas spp. were identified and virulence quantified using an animal model. Virulence was measured by determining a 50% lethal dose (LD₅₀) 43 h after oral administration of live bacteria. The LD₅₀ of virulent Aeromonas isolates ranged from log₁₀ 7.53 (mean) organisms to log₁₀ 8.88 (mean). Some isolates were avirulent in this model. Detection of cytotoxic activity in culture supernatants correlated with virulence (Fisher exact test, P = 0.0029). There was no correlation between LD₅₀ and the source of the isolate, β-haemolysis or lipopolysaccharide (LPS) banding profile on SDS-PAGE. In this animal model, virulence was multifactorial in that: (i) bacterial multiplication in the gut was associated with fatal infection; (ii) the increase in bacterial numbers in the gut of mice administered a lethal dose of bacteria was accompanied by accumulation of fluid; and (iii) there was evidence of extraintestinal spread of infection. Protection of suckling mice by rabbit antiserum to Aeromonas cell envelopes was observed.     

Insecticide susceptibility and resistance of Blattella germanica (Blattaria: Blattellidae) in Seoul, Republic of Korea, 2007

The susceptibility of Blattella germanica (L.) in the Republic of Korea (ROK) to insecticides was evaluated under laboratory conditions using 12 insecticides currently used by the local public health centers and/or pest control operators in the ROK. The insecticides included seven pyrethroids and five organophosphates. Based on their LD₅₀ values, the order of susceptibility of B. germanica adults to the insecticides was chlorpyrifos-methyl, profenofos and chlorpyrifos with values of 0.07, 0.29 and 0.88 µg/♀, respectively. The least susceptibility was obtained with tetramethrin at LD₅₀ of 7.39 µg/♀. In the comparative resistance test, the resistance ratios (RR) of 12 insecticides were compared to each other using field-collected B. germanica adults in Seoul between 1993 and 2007. Blattella germanica demonstrated higher RRs to pyrethroids such as λ-cyhalothrin, and low RRs among the organophosphates. Among the pyrethroids, λ-cyhalothrin had the highest RRs of 111- and 129-fold differences at LD₅₀ and LD₉₀ values, respectively. Among the organophosphates, profenofos was observed to have the highest RRs of 4- and 15-fold differences at LD₅₀ and LD₉₀ values, respectively. However, there were no significant differences in susceptibility to tetramethrin, chlorpyrifos and fenitrothion. Blattella germanica was more susceptible to pyridafenthion showing a 0.7-fold difference in a resistance ratio (RRLD₅₀= LD₅₀ value of 2007/LD₅₀ value of 1993). Resistance ratio of tetramethrin was low, but susceptibility was also not high.     

The chick embryo test agrees with the mouse bio-assay for assessment of the pathogenicity of Listeria species

The pathogenicity of 20 strains of Listeria was determined with the mouse intravenous bio-assay and the 10-day chick embryo chorioallontoic membrane test. In the former, survival in the spleen was measured and in the latter, the LD₁₀₀ and LD₅₀. There was good correlation between the results of the two tests. Listeria monocytogenes strains that grow in the mouse spleen had an LD₁₀₀ of < 125 organisms, while strains in which the numbers of organisms in the mouse spleen remain constant had an LD₁₀₀ > 125 organisms. Listeria seeligeri, L. innocua, L. welshimeri, L. grayi and L. murrayi did not persist in the spleen and the numbers of organisms used did not kill chick embryos.     

The effects of interleukin 4 on the cell cycle of a human B-cell lymphoma line

Abstract. Exposure of Farage, a human B-cell lymphoma line, to IL-4 for 3–11 days led to inhibition of tritiated thymidine ([³H]dT) uptake by the cells. Study of the incorporation of 5-bromodeoxyuridine by Farage cells showed that IL-4 reduced significantly the number of cells in the S phase of the cell cycle and increased the proportion of cells in the G₁ phase. Limiting dilution analysis of proliferation demonstrated that IL-4 decreased the frequency of clone-forming cells by 40%. IL-4 did not reduce the viability of Farage cells. On the contrary, IL-4 diminished the spontaneous death of Farage cells in culture, as determined by pulse chase analysis of cells which were labelled with [³H]dT. Moreover, the pre-treatment of Farage cells with IL-4 prevented their death induced by exposure to a high dose of staurosporine. IL-4 abrogated the staurosporine-induced arrest of cells in the G₂+ M phase and replaced it by accumulation of cells in the G₁ phase. IL-4 protected Farage cells from the radioactive suicide caused by the uptake of [³H]dT by dividing cells. The cytokine failed to prevent the damage to Farage cells exerted by mitomycin C, which affected cellular DNA regardless of the phase of the cell cycle. The data obtained showed that IL-4 inhibited the division of B cells by arresting their progression through the early stages of the cell cycle. This inhibition of the cell efflux from G₁ phase plays an important role in the protection against cell death during further stages of the cell cycle.     

Evaluation of the chick embryo for the determination of relative virulence of Neisseria meningitidis

Abstract The chick embryo model was evaluated as a method to compare virulence between selected strains of Neisseria meningitidis . Inoculation of 13-day-chick embryos via the egg yolk distinguished strains having an LD₅₀ of 10³ colony forming units (CFU) or greater (low virulence) from those having an LD₅₀ of approximately 10¹ or less (high virulence). A strain of serogroup B and a spontaneous nonpiliated strain of group C were found to be of relatively high virulence while a strain of N. lactamica , a serogroup A carrier strain, and certain nongroupable strains were found to be of low virulence. Strains having an LD₅₀ of 10² were not differentiated from either of these. Alternatively, inoculation of the chorioallantoic membrane (CAM) of 9-day-old chick embryos statistically differentiated most strains of N. meningitidis although inoculation via this route was less sensitive.     

SPERMATOGONIAL STEM CELL RENEWAL IN THE MOUSE: II AFTER CELL LOSS

The repair of the mouse seminiferous epithelium after cell loss has been studied in seminiferous tubules mounted in toto . Cell loss was inflicted by injection of Myleran in a dose of 10 mg/kg body weight. In stages 7–8, in which we mainly counted, the numbers of A_isolated (A_is), A_paired (A_pr), A_aligned (A_al) and A₁ spermatogonia and resting primary spermatocytes decreased after injection. After about 24 days normal numbers of A₁ spermatogonia were found again. Thereafter a substantial overshoot in the number of A₁ spermatogonia was found.
While normally most of the A_pr and A_al cells differentiate into A₁ spermatogonia in stages 3 and 4 and do not divide until stage 9, during repair they pass through one more division during stages 6 and 7. Normally, during these stages divisions of these spermatogonia are rare. Owing to this extra division the transformation of A_pr and A_al into A₁ spermatogonia is delayed from stage 3 or 4 to stage 8, i.e. still before stage 9, in which A₁ spermatogonia divide. From 16 days after the injection onwards the extra division takes place less generally and more and more cells transform into A₁ spermatogonia at the normal time.     

Dopamine Neurotoxicity: Inhibition of Mitochondrial Respiration

Abstract: Dopamine, due to metabolism by monoamine oxidase or autoxidation, can generate toxic products such as hydrogen peroxide, oxygen-derived radicals, semiquinones, and quinones and thus exert its neurotoxic effects. Intracerebroventricular injection of dopamine into rats pretreated with the monoamine oxidase nonselective inhibitor pargyline caused mortality in a dose-dependent manner with LD₅₀ = 90 µg. Norepinephrine was less effective with LD₅₀ = 141 µg. The iron chelator desferrioxamine completely protected against dopamine-induced mortality. In the absence of pargyline more rats survived, indicating that the products of dopamine enzymatic metabolism are not the main contributors to dopamine-induced toxicity. Biochemical analysis of frontal cortex and striatum from rats that received a lethal dose of dopamine did not show any difference from control rats in lipid and protein peroxidation and glutathione reductase and peroxidase activities. Moreover, dopamine significantly reduced the formation of iron-induced malondialdehyde in vitro, thus suggesting that earlier events in cell damage are involved in dopamine toxicity. Indeed, dopamine inhibited mitochondrial NADH dehydrogenase activity with IC₅₀ = 8 µ M , and that of norepinephrine was twice as much (IC₅₀ = 15 µ M ). Dopamine-induced inhibition of NADH dehydrogenase activity was only partially reversed by desferrioxamine, which had no effect on norepinephrine-induced inhibition. These results suggest that catecholamines can cause toxicity not only by inducing an oxidative stress state but also possibly through direct interaction with the mitochondrial electron transport system. The latter was further supported by the ability of ADP to reverse dopamine-induced inhibition of NADH dehydrogenase activity in a dose-dependent manner.     

Outward-rectifying K+ channel activities regulate cell elongation and cell division of tobacco BY-2 cells

Potassium ions (K⁺) are required for plant growth and development, including cell division and cell elongation/expansion, which are mediated by the K⁺ transport system. In this study, we investigated the role of K⁺ in cell division using tobacco BY-2 protoplast cultures. Gene expression analysis revealed induction of the Shaker -like outward K⁺ channel gene, NTORK1 , under cell-division conditions, whereas the inward K⁺ channel genes NKT1 and NtKC1 were induced under both cell-elongation and cell-division conditions. Repression of NTORK1 gene expression by expression of its antisense construct repressed cell division but accelerated cell elongation even under conditions promoting cell division. A decrease in the K⁺ content of cells and cellular osmotic pressure in dividing cells suggested that an increase in cell osmotic pressure by K⁺ uptake is not required for cell division. In contrast, K⁺ depletion, which reduced cell-division activity, decreased cytoplasmic pH as monitored using a fluorescent pH indicator, SNARF-1. Application of K⁺ or the cytoplasmic alkalizing reagent (NH₄)₂SO₄ increased cytoplasmic pH and suppressed the reduction in cell-division activity. These results suggest that the K⁺ taken up into cells is used to regulate cytoplasmic pH during cell division.     

Baseline toxicity of emamectin and spinosad to Helicoverpa armigera (Lepidoptera: Noctuidae) for resistance monitoring

Acute toxicity studies of emamectin and spinosad against Helicoverpa armigera revealed that the pest is highly susceptible to both the insecticides. The median lethal dose (LD₅₀) of emamectin is 3.86 × 10⁻³ µg per larva. The median lethal concentrations (LC₅₀) of emamectin and spinosad were found to be 0.09 and 2.94 ppm, respectively. The discriminating doses were fixed based on the LC₉₅ of the susceptible population of H. armigera as 0.80 ppm for emamectin and 10 ppm for spinosad. Resistance was not observed when the discriminating doses of emamectin and spinosad were applied on field-collected populations of H. armigera from two intensive cotton growing areas, Coimbatore and Madurai, India.     

Diel rhythmicity in amino acid uptake by Prochlorococcus

The marine cyanobacterium Prochlorococcus , the most abundant phototrophic organism on Earth, numerically dominates the phytoplankton in nitrogen (N)-depleted oceanic gyres. Alongside inorganic N sources such as nitrite and ammonium, natural populations of this genus also acquire organic N, specifically amino acids. Here, we investigated using isotopic tracer and flow cytometric cell sorting techniques whether amino acid uptake by Prochlorococcus is subject to a diel rhythmicity, and if so, whether this was linked to a specific cell cycle stage. We observed, in contrast to diurnally similar methionine uptake rates by Synechococcus cells, obvious diurnal rhythms in methionine uptake by Prochlorococcus cells in the tropical Atlantic. These rhythms were confirmed using reproducible cyclostat experiments with a light-synchronized axenic Prochlorococcus (PCC9511 strain) culture and ³⁵S-methionine and ³H-leucine tracers. Cells acquired the tracers at lower rates around dawn and higher rates around dusk despite >10⁴ times higher concentration of ammonium in the medium, presumably because amino acids can be directly incorporated into protein. Leucine uptake rates by cells in the S+G₂ cell cycle stage were consistently 2.2 times higher than those of cells at the G₁ stage. Furthermore, S+G₂ cells upregulated amino acid uptake 3.5 times from dawn to dusk to boost protein synthesis prior to cell division. Because Prochlorococcus populations can account from 13% at midday to 42% at dusk of total microbial uptake of methionine and probably of other amino acids in N-depleted oceanic waters, this genus exerts diurnally variable, strong competitive pressure on other bacterioplankton populations.     

Toxicity of six commercially formulated insecticides and biopesticides to third instar larvae of mushroom sciarid, Lycoriella ingenua Dufour (Diptera: Sciaridae), in New South Wales, Australia

Abstract  Lycoriella ingenua is one of the major pests of cultivated mushrooms, Agaricus bisporus (Lange) Imbach . Insecticide resistance among mushroom sciarid populations has been reported from other countries, and there is a need to determine the toxicity of currently approved and potential pesticides to sustain control of mushroom sciarid populations in Australia. The present study investigated the toxicity of six commercial formulations of insecticides or biopesticides against third instar larvae of L .  ingenua using laboratory bioassays. Insecticide treatments were incorporated into the growing medium for sciarid larvae and the concentration of the pesticide, which killed 90% of the test population (LD₉₀) determined the efficacy of selected insecticides. Triflumuron was the most effective insecticide against L. ingenua with an LD₉₀ of 53.12 mg active ingredient (a.i.)/m² followed by cyromazine (LD₉₀, 179.68 mg a.i./m²) and diazinon (LD₉₀, 261.72 mg a.i./m²). Abamectin and Bacillus thuringiensis ssp. israelensis were ineffective against L. ingenua . Steinernema feltiae , an entomopathogenic nematode, reduced the number of third instar larvae of L. ingenua only when applied at a higher rate (LD₉₀, 732 422 nematodes/m²) than was recommended on the label.     

Net fluxes and pools of nitrogenous compounds during suspension culture of photoautotrophic Chenopodium rubrum cells

Abstract. Suspension cultured cells of Chenopodium rubrum were grown photoautotrophically under a diurnal light-dark cycle of 16-8h. The following phases of the batch culture were differentiated: a short lag, a cell division phase terminated by a pronounced transition to stationary maintenance which finally gradually passed into senescence. Nitrogen fluxes typical of these stages were followed by measuring uptake of NO₃ and NH₄⁺ from the medium and their incorporation into the cellular fractions of nitrogenous compounds. Activities of seven N-metabolizing enzymes were determined. Compartmentation of enzymes and nitrogenous compounds was analysed after isolation of intact chloroplasts and vacuoles from protoplasts. Eighty-two per cent of the N originally present in the medium was taken up and incorporated to an extent of 80% into protein until the end of the division phase. Net protein synthesis ceased upon transition to the stationary phase. During the division phase a vacuolar pool of NO₃ was established and then maintained throughout the resting phase. Free cellular NH₄⁺ was not localized within the vacuole and responded to the ammonium content of the medium. Amino acids accumulated in the cells especially during the stationary phase, during which they were present in the vacuole. Typical nitrogen relations are portrayed as flux diagrams for one day of each of the essential developmental phases. The enzyme activities were easily sufficient to account for the observed flow rates of the corresponding nitrogenous compounds. Hence, uptake of NO₃ and NH₄⁺ must be considered as steps limiting N metabolism in Chenopodium rubrum cell suspensions.     

In Vitro Effect of Tetanus Toxin on GAB A Release from Rat Hippocampal Slices

Abstract Ca²⁺-dependent K⁺-stimulated γ-aminobutyric acid release from rat hippocampal slices was reduced about 30% by pre-incubation of the slices with 10⁴ mouse LD₅₀/ml tetanus toxin for 3 h at 37°C.     

The effect of acute X-irradiation on the development of the plaice Pleuronectes platessa L.

Plaice eggs were irradiated with 80 kV peak X-rays at 5 % of their development from fertilization to hatching. The range of doses used was approximately 30–150 rads and the doses were measured with thermoluminescent dosimeters. The percentage hatch of eggs, survival of 3-week old larvae and survival to metamorphosis were noted. The results show that at one of the most radiosensitive stages of development, the LD₅₀/metamorphosis for plaice is approximately 90 rads. There is a steep response curve where a dose of 30 rads has little effect and where doses above 150 rads kill the majority of the larvae before metamorphosis.     

Opsonic and protective activity of five human IgM monoclonal antibodies reactive with lipopolysaccharide antigen of Pseudomonas aeruginosa

Abstract International Antigen Typing Schema (IATS) serotypes 1, 2, 5, 6, 8 and 11 comprise approximately vn80% of Pseudomonas aeruginosa strains isolated from blood, wounds and respiratory specimens. Five human immunolgobulin M (IgM) monoclonal antibodies (MAbs) reactive with lipopolysaccharide O antigens of these IATS serotypes were studies in an opsonophagocytic assay. The assay employed human polymorphonuclear leukocytes, 2% guinea pig serum as the complement source and MAb. Each MAb promoted killing of inoculum of the homologous LPS serotype. The opsonic activity of each MAb was complement-dependent. In a murine model of Pseudomonas burn wound sepsis the LD₅₀ of five strains of P. aeuruginosa was increased ≥ 22-fold by MAb-treatment (1.0 mg/kg). The mean effective dose of the five MA0bs in mice challenged with approximately 10 LD₅₀ of the homologous LPS serotype ranged from < 0.01 mg/kg to 1.00 mg/kg.     

EFFECTS OF ACTINOMYCIN D AND PUROMYCIN ON THE CELL PROGRESS FROM M TO G1 AND S STAGES IN CULTURED MOUSE LEUKEMIA L5178Y CELLS

Actinomycin D (0.5 μg/ml) did not prevent M stage cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The position of the block was approximately 1.4 hr before S stage or just after the beginning of G₁ stage. Actinomycin D in this concentration also significantly depressed uridine-³H uptake into G₁ stage cells, but did not suppress leucine-³H uptake by M and G₁ cells. This suggests that some proteins may be synthesized in M and G₁ stage cells by messenger RNA left over from the previous cell cycle. However, entry of G₁ cells into S stage would require synthesis of new messenger RNA near the beginning of G₁ stage. Puromycin (10 μg/ml) did not prevent M cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The site of blockage was about 0.7 hr before S stage or in the first two-third of G₁ stage. This might be the site where the cells synthesize new G₁ proteins necessary for entry to S stage.
Comparison of sensitivities of G₁ and G₂ stages to the two antibiotics reveals that the puromycin sensitivity of G₁ cells was similar to that of G₂ cells, but the actinomycin D sensitivity of G₁ was greater than that of G₂ cells.     

Tn5-induced Xenorhabdus bovienii lecithinase mutants demonstrate reduced virulence for Galleria mellonella larvae

A derivative of Tn5 was used to construct a variety of stable insertion mutations in the entomopathogenic bacterium, Xenorhabdus bovienii T228/1. Mutants which had altered expression of Congo Red binding ability, ampicillin resistance, haemolytic activity and lecithinase were isolated. Isolates with altered lecithinase activity had either lost ability to produce this enzyme or showed reduced expression. The role of lecithinase in pathogenesis of X. bovienii T228/1 for Galleria mellonella larvae was examined by LD₅₀ analysis. Maximum killing of G. mellonella was observed at 72 h post infection for both the wild-type parent strain and a lecithinase mutant 34(45). However, the LD₅₀ value for the wild-type parent strain (8·7 cells per larva) was significantly less than that calculated for the lecithinase mutant (35·5 cells per larva). The data suggested that although lecithinase is a virulence factor produced by X. bovienii , lecithinase activity alone is not sufficient for killing of G. mellonella larvae.     

Respiration, cyanide-insensitive oxygen uptake and oxidative phosphorylation in cyanobacteria

Cyanide-insensitive oxygen uptake in the dark of 9 species of cyanobacteria was 6–20% of the total oxygen uptake of intact cells. In Phormidium , no cyanideinsensitive oxygen uptake was observed. In intact cells, the I₅₀ value for cyanide was significantly lower in cyanobacteria of the taxonomic sections I to III (1–9 μ M ) than in those from section IV and V (10–60 μ M ). Cyanide-insensitive oxygen uptake in the cell-free system of Anabaena variabilis was not affected by typical inhibitors of the alternative pathway of plants. Cell-free oxidation of cytochrome c was completely inhibited by cyanide with an I₅₀ value of 0.5–1 μ M . Electron transport of intact cells without cyanide present yielded P/O ratios of 0.7–3.0. The data on oxidative phosphorylation using intact cells and the cell-free system, indicate that cyanide-insensitive oxygen uptake is not coupled to ATP formation.     

Peroxide inducible phage reactivation in Bacteroides fragilis

Abstract Reactivation of UV-irradiated phage b-1 was induced by H₂O₂ and UV in Bacteroides fragilis . The characteristics of H₂O₂ and UV induced phage reactivation differ from a previously reported oxygen induced reactivation system. The survival of B. fragilis cells after UV irradiation was also increased by pretreatment with H₂O₂. DNA synthesis was not inhibited in the host cells exposed to H₂O₂ concentrations which induced phage reactivation. The pattern of DNA degradation and synthesis after UV irradiation with and without H₂O₂ differed from the effect of O₂ on DNA synthesis in irradiated B. fragilis cells.