Rapid detection of viable Legionella pneumophila in tap water by a qPCR and RT‐PCR‐based method

Aims

A molecular method for a rapid detection of viable Legionella pneumophila of all serogroups in tap water samples was developed as an alternative to the reference method (ISO). Legionellae are responsible for Legionnaires’ disease, a severe pneumonia in humans with high lethality.

Methods and Results

The developed method is based on a nutritional stimulation and detection of an increase in precursor 16S rRNA as an indicator for viability. For quantification, DNA was detected by qPCR. This method was compared to the ISO method using water samples obtained from public sports facilities in Switzerland. The sensitivity and specificity were 91 and 97%, respectively, when testing samples for compliance with a microbiological criterion of 1000 cell equivalents per l.

Conclusion

The new method is sensitive and specific for Leg. pneumophila and allows results to be obtained within 8 h upon arrival, compared to one week or more by the ISO method.

Significance and Impact of the Study

The method represents a useful tool for a rapid detection of viable Leg. pneumophila of all serogroups in water by molecular biology. It can be used as an alternative to the ISO method for official water analysis for legionellae and particularly when a short test time is required.     

Treatment of Grape Berries,a Nonclimacteric Fruit with a Synthetic Auxin,Retards Ripening and Alters the Expression of Developmentally Regulated Genes

Treatment of grape (Vitis vinifera L.) berries with the synthetic auxin-like compound benzothiazole-2-oxyacetic acid (BTOA) caused a delay in the onset of ripening of approximately 2 weeks. This was manifested as a retardation of the increases in berry weight, color, deformability, and hexose concentration. BTOA treatment also delayed by 2 weeks the increase in abscisic acid level that normally accompanies ripening and altered the expression of a number of developmentally regulated genes. A putative vacuolar invertase, which is normally expressed from berry set until ripening and turned off after ripening commences, remained expressed throughout development in BTOA-treated grape berries. This elevated expression resulted in increased levels of invertase activity. In contrast, the up-regulation of four other genes normally switched on at the time of ripening was delayed in BTOA-treated fruit. These included chalcone synthase and UDP-glucose-flavonoid 3-O-glucosyl transferase, both of which are involved in anthocyanin synthesis, a chitinase, and a ripening-related gene of an unknown function. These observations support the view that auxins (perhaps in conjunction with abscisic acid) may have a role in the control of grape berry ripening by affecting the expression of genes involved in the ripening process.     

Inositol Trisphosphate Metabolism in Carrot (Daucus carota L.) Cells

The metabolism of exogenously added d-myo-[1-³H]inositol 1,4,5-trisphosphate (IP₃) has been examined in microsomal membrane and soluble fractions of carrot (Daucus carota L.) cells grown in suspension culture. When [³H]IP₃ was added to a microsomal membrane fraction, [³H]IP₂ was the primary metabolite consisting of approximately 83% of the total recovered [³H] by paper electrophoresis. [³H]IP was only 6% of the [³H] recovered, and 10% of the [³H]IP₃ was not further metabolized. In contrast, when [³H]IP₃ was added to the soluble fraction, approximately equal amounts of [³H]IP₂ and [³H]IP were recovered. Ca²⁺ (100 micromolar) tended to enhance IP₃ dephosphorylation but inhibited the IP₂ dephosphorylation in the soluble fraction by about 20%. MoO₄²⁻ (1 millimolar) inhibited the dephosphorylation of IP₃ by the microsomal fraction and the dephosphorylation of IP₂ by the soluble fraction. MoO₄²⁻, however, did not inhibit the dephosphorylation of IP₃ by the soluble fraction. Li⁺ (10 and 50 millimolar) had no effect on IP₃ metabolism in either the soluble or membrane fraction; however, Li⁺ (50 millimolar) inhibited IP₂ dephosphorylation in the soluble fraction about 25%.     

The effect of fluorescent labeling on calcium-induced fusion of fusogenic carrot protoplasts

Various fluorescent compounds — carboxyfluorescein, scopoletin, fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC), rhodamine 123, and rhodamine B ethyl ester — were used to study their effects on calcium-induced fusion of fusogenic carrot (Daucus carota L.) protoplasts. These protoplasts normally fused at a high percentage (50–60%) in response to 10 mM calcium, pH 6.0; however, if cells had been labeled with scopoletin, FITC, or RITC, fusion was greatly reduced. In contrast, labeling with carboxyfluorescein, rhodamine 123, or rhodamine B ethyl ester had no detectable effect on calcium-induced fusion. The two rhodamine dyes are shown to be localized in mitochondria.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - FITC fluorescein isothiocyanate - RITC rhodamine isothiocyanate - PE phosphatidylethanolamine     

Age-dependency of lymphocyte ecto-5'-nucleotidase activity

The activity of lymphocyte ecto-5'-nucleotidase (ecto-5'-NT) decreases with advancing age, T lymphocyte ecto-5'-NT activity begins to fall after the age of 40 and subjects in the 41 to 50, 51 to 60, 61 to 75, and 75 to 85 age ranges of life have 57, 52, 38, and 19%, respectively, of the T lymphocyte ecto-5'-NT activity of subjects under the age of 40. TG cells (suppressor cells) have 39% of the ecto-5'-NT activity of Tnon-G cells (helper cells) but the increase in numbers of TG cells that occurs with age explains only about 14% of the age-related fall in T lymphocyte ecto-5'-NT activity. B lymphocyte ecto-5'-NT activity remains stable until age 60 and subjects over 60 years of age have 42% of the B lymphocyte ecto-5'-NT activity of subjects under age 60.     

Purine deoxynucleosides and adenosine dialdehyde decrease 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis in cultured T and B lymphoblasts.

Deoxyadenosine (dAdo) and deoxyguanosine (dGuo) decrease methionine synthesis from homocysteine in cultured lymphoblasts; because of the possible trapping of 5-methyltetrahydrofolate this could lead to decreased purine nucleotide synthesis. Since purine deoxynucleosides could also inhibit purine synthesis de novo at an early step not involving folate metabolism, we measured in azaserine-treated cells 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis using [14C]formate. In the T lymphoblasts, Z-base-dependent purine nucleotide synthesis was decreased 26% by 0.3 microM-dAdo, 21% by 1 microM-dGuo and 28% by 1 microM-adenosine dialdehyde, a potent S-adenosylhomocysteine hydrolase inhibitor; homocysteine fully reversed the inhibitions. The B lymphoblasts were considerably less sensitive to the deoxynucleoside-induced decrease in Z-base-dependent purine nucleotide synthesis, with 100 microM-dAdo required for significant inhibition and no inhibition by dGuo at this concentration; homocysteine partly reversed the inhibition by dAdo. The observed decrease in Z-base-dependent purine nucleotide synthesis could not be attributed either to dUMP depletion changing the folate pools or to decreased ATP availability because dUrd was without effect and during the experimental period the intracellular ATP concentration did not change significantly. Cells with 5,10-methylenetetrahydrofolate reductase deficiency were relatively resistant to inhibition of Z-base-dependent purine nucleotide synthesis by dAdo and adenosine dialdehyde. Our results suggest that deoxynucleosides decrease purine nucleotide synthesis by trapping 5-methyltetrahydrofolate.     

An angiotensin II antagonist with strongly prolonged action

A highly hydrophobic analogue of angiotensin II (AT), [Sar1,(2',3',4',5',6'-Br5)Phe8]AT exhibited strong and persistent specific antagonism against AT, both in vitro and in vivo. This peptide exhibited 32% of the binding affinity of [Sar1]AT towards membranes of bovine adrenal cortex, it was a specific AT antagonist of irreversible character on smooth muscle assays, and it also suppressed for over 120 min at 7.10(-8) M/kg the blood pressure response towards AT in the rat blood pressure assay. This compound harbours therefore the potential of a new class of AT-specific antihypotensive drugs.