Chemical synthesis of (S)-4,5-dihydroxy-2,3-pentanedione, a bacterial signal molecule precursor, and validation of its activity in Salmonella typhimurium

We describe an original, short, and convenient chemical synthesis of enantiopure (S)-4,5-dihydroxy-2,3-pentanedione (DPD), starting from commercial methyl (S)-(-)-2,2-dimethyl-1,3-dioxolane-4-carboxylate. DPD is the precursor of autoinducer (AI)-2, the proposed signal for bacterial interspecies communication. AI-2 is synthesized by many bacterial species in three enzymatic steps. The last step, a LuxS-catalyzed reaction, leads to the formation of DPD, which spontaneously cyclizes into AI-2. AI-2-like activity of the synthesized molecule was ascertained by the Vibrio harveyi bioassay. To further validate the biological activity of synthetic DPD and to explore its potential in studying DPD (AI-2)-mediated signaling, a Salmonella typhimurium luxS mutant was constructed. Expression of the AI-2 regulated lsr operon can be rescued in this luxS mutant by addition of synthetic DPD or genetic complementation. Biofilm formation by S. typhimurium has been reported to be defective in a luxS mutant, and this was confirmed in this study to test DPD for chemical complementation. However, biofilm formation of the luxS mutant cannot be restored by addition of DPD. In contrast, introduction of luxS under control of its own promoter complemented biofilm formation. Further results demonstrated that biofilm formation of the luxS mutant cannot be restored with luxS under control of the strong nptII promoter. This indicates that altering the intrinsic promoter activity of luxS affects Salmonella biofilm formation. Conclusively, we synthesized biologically active DPD. Using this chemical compound in combination with genetic approaches opens new avenues in studying AI-2-mediated signaling.     

Fission yeast homologue of Tip41-like proteins regulates type 2A phosphatases and responses to nitrogen sources

A fission yeast (Schizosaccharomyces pombe) gene encoding a member of the TIP41-like protein family was identified and characterized. Deletion of the fission yeast tip41 gene leads to slower growth when ammonium chloride is the nitrogen source, but the growth rate is not affected when adenine is the nitrogen source. The tip41 mutant cells also enter the G1 phase of the cell cycle earlier than wild-type cells in response to nitrogen starvation. Overexpression of tip41(+) causes cell death, spherical cell morphology and blocks the shift to G1 phase upon nitrogen starvation. Overexpression of tip41(+) increases the activity of type 2A phosphatase. In a ppa2 deletion strain with reduced PP2A activity, overexpression of tip41(+) no longer blocks the shift to G1 upon nitrogen starvation. These results suggest that fission yeast Tip41 plays a role in cellular responses to nitrogen nutrient conditions at least partly through regulation of type 2A phosphatase activity.     

Identification of conserved domains in Salmonella muenchen flagellin that are essential for its ability to activate TLR5 and to induce an inflammatory response in vitro

The bacterial surface protein flagellin is widely distributed and well conserved among distant bacterial species. We and other investigators have reported recently that purified flagellin from Salmonella dublin or recombinant flagellin of Salmonella muenchen origin binds to the eukaryotic toll receptor TLR5 and activates the nuclear translocation of NF-kappaB and mitogen-activated protein kinase, resulting in the release of a host of pro-inflammatory mediators in vitro and in vivo. The amino acid sequence alignment of flagellins from various Gram-negative bacteria shows that the C and N termini are well conserved. It is possible that sequences within the N and C termini or both may regulate the pro-inflammatory activity of flagellin. Here we set out to map more precisely the regions in both termini that are required for TLR5 activation and pro-inflammatory signaling. Systematic deletion of amino acids from either terminus progressively reduced eukaryotic pro-inflammatory activation. However, deletion of amino acids 95-108 (motif N) in the N terminus and 441-449 (motif C) in the C terminus abolished pro-inflammatory activity completely. Site-directed mutagenesis analysis provided further evidence for the importance of motifs N and C. We also present evidence for the functional role of motifs N and C with the TLR5 receptor using a reporter assay system. Taken together, our results demonstrate that the pro-inflammatory activity of flagellin results from the interaction of motif N with the TLR5 receptor on the cell surface.     

Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle

The effects of thymol on steps of excitation-contraction coupling were studied on fast-twitch muscles of rodents. Thymol was found to increase the depolarization-induced release of calcium from the sarcoplasmic reticulum, which could not be attributed to a decreased calcium-dependent inactivation of calcium release channels/ryanodine receptors or altered intramembrane charge movement, but rather to a more efficient coupling of depolarization to channel opening. Thymol increased ryanodine binding to heavy sarcoplasmic reticulum vesicles, with a half-activating concentration of 144 micro M and a Hill coefficient of 1.89, and the open probability of the isolated and reconstituted ryanodine receptors, from 0.09 +/- 0.03 to 0.22 +/- 0.04 at 30 micro M. At higher concentrations the drug induced long-lasting open events on a full conducting state. Elementary calcium release events imaged using laser scanning confocal microscopy in the line-scan mode were reduced in size, 0.92 +/- 0.01 vs. 0.70 +/- 0.01, but increased in duration, 56 +/- 1 vs. 79 +/- 1 ms, by 30 micro M thymol, with an increase in the relative proportion of lone embers. Higher concentrations favored long events, resembling embers in control, with duration often exceeding 500 ms. These findings provide direct experimental evidence that the opening of a single release channel will generate an ember, rather than a spark, in mammalian skeletal muscle.     

Specificity of the tumor necrosis factor-induced protein 6-mediated heavy chain transfer from inter-alpha-trypsin inhibitor to hyaluronan: implications for the assembly of the cumulus extracellular matrix

The formation of the hyaluronan-rich cumulus extracellular matrix is crucial for female fertility and accompanied by a transesterification reaction in which the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI)-related proteins are covalently transferred to hyaluronan. Tumor necrosis factor-induced protein-6 (TNFIP6) is essential for this transfer reaction. Female mice deficient in TNFIP6 are infertile due to the lack of a correctly formed cumulus matrix. In this report, we characterize the specificity of TNFIP6-mediated HC transfer from IalphaI to hyaluronan. Hyaluronan oligosaccharides with eight or more monosaccharide units are potent acceptors in the HC transfer, with longer oligosaccharides being somewhat more efficient. Epimerization of the N-acetyl-glucosamine residues to N-acetyl-galactosamines (i.e. in chondroitin) still allows the HC transfer although at a significantly lower efficiency. Sulfation of the N-acetyl-galactosamines in dermatan-4-sulfate or chondroitin-6-sulfate prevents the HC transfer. Hyaluronan oligosaccharides disperse cumulus cells from expanding cumulus cell-oocyte complexes with the same size specificity as their HC acceptor specificity. This process is accompanied by the loss of hyaluronan-linked HCs from the cumulus matrix and the appearance of oligosaccharide-linked HCs in the culture medium. Chondroitin interferes with the expansion of cumulus cell-oocyte complexes only when added with exogenous TNFIP6 before endogenous hyaluronan synthesis starts, supporting that chondroitin is a weaker HC acceptor than hyaluronan. Our data indicate that TNFIP6-mediated HC transfer to hyaluronan is a prerequisite for the correct cumulus matrix assembly and hyaluronan oligosaccharides and chondroitin interfere with this assembly by capturing the HCs of the IalphaI-related proteins.     

Single neonatal treatment with beta-endorphin (hormonal imprinting) extremely enhances nocistatin level of cerebrospinal fluid in adult rats

In earlier experiments endorphin treatment of newborn rats caused the decrease of brain serotonin content, increasing aggressivity, enhanced sexual activity of females and changes in the binding capacity of uterine estrogen receptors at adult age, however nociceptin content of the cerebrospinal fluid was not changed. In the present experiment neonatal treatment of male and female rats was done with a single dose of 3 microg beta-endorphin and in five months old rats the level of nociceptin antagonist nocistatin was determined by radioimmunoassay in the cerebrospinal fluid. In both genders the amount of nocistatin was one magnitude higher in the endorphin treated groups. There was also a significant difference between the male and female nocistatin level in the treated and non-treated groups alike, with the advantage of females. The results call attention to the possibility of influencing pain-tolerance for life, by the pain-provoked endorphin levels during delivery.     

Effect of thymol on calcium handling in mammalian ventricular myocardium

Concentration-dependent effects of thymol on calcium handling were studied in canine and guinea pig cardiac preparations (Langendorff-perfused guinea pig hearts, canine ventricular trabeculae, canine sarcoplasmic reticular vesicles and single ryanodine receptors). Thymol induced a concentration-dependent negative inotropic action in both canine and guinea pig preparations (EC(50) = 297 +/- 12 microM in dog). However, low concentrations of thymol reduced intracellular calcium transients in guinea pig hearts without decreasing contractility. At higher concentrations both calcium transients and contractions were suppressed. In canine sarcoplasmic reticular vesicles thymol induced rapid release of calcium (V(max) = 0.47 +/- 0.04 nmol s(-1), EC(50) = 258 +/- 21 microM, Hill coefficient = 3.0 +/- 0.54), and decreased the activity of the calcium pump (EC(50) = 253 +/- 4.7 microM, Hill coefficient = 1.62 +/- 0.05). Due to the less sharp concentration-dependence of the ATPase inhibition, this effect was significant from 50 microM, whereas the thymol-induced calcium release only from 100 microM. In single ryanodine receptors incorporated into artificial lipid bilayer thymol induced long lasting openings, having mean open times increased with 3 orders of magnitude, however, the specific conductance of the channel remained unaltered. This effect of thymol was not voltage-dependent and failed to prevent the binding of ryanodine. In conclusion, the negative inotropic action of thymol can be explained by reduction in calcium content of the sarcoplasmic reticulum due to the combination of the thymol-induced calcium release and inhibition of the calcium pump. The calcium-sensitizer effect, observed at lower thymol concentrations, indicates that thymol is likely to interact with the contractile machinery also.     

Effect of the sesterterpene-type metabolites, ophiobolins A and B, on zygomycetes fungi

Ophiobolins are sesterterpene-type phytotoxins produced by fungi belonging mainly to the genus Bipolaris. In this study, the antifungal effect of ophiobolins A and B on different zygomycetes has been examined. Depending on the zygomycete tested, MIC values of 3.175-50 μg mL(-1) were found for ophiobolin A and 25-50 μg mL(-1) for ophiobolin B. Ophiobolin A inhibited sporangiospore germination of Mucor circinelloides and caused morphological changes; the fungus formed degenerated, thick or swollen cells with septa. Cytoplasm effusions from the damaged cells were also observed. Fluorescence microscopy after annexin and propidium iodide staining of the treated cells suggested that the drug induced an apoptosis-like cell death process in the fungus.     

Changing climate in Hungary and trends in the annual number of heat stress days

Global climate change can have serious direct effects on animal health and production through heat stress. In Hungary, the number of heat stress days per year (YNHD), i.e., days when the temperature humidity index (THI) is above a specific comfort threshold, has increased in recent years based on observed meteorological data. Between 1973 and 2008, the countrywide average increase in YNHD was 4.1% per year. Climate scenarios based on regional climate models (RCM) were used to predict possible changes in YNHD for the near future (2021–2050) relative to the reference period (1961–1990). This comparison shows that, in Hungary, the 30-year mean of YNHD is expected to increase by between 1 and 27 days, depending on the RCM used. Half of the scenarios investigated in this study predicted that, in large parts of Hungary, YNHD will increase by at least 1 week. However, the increase observed in the past, and that predicted for the near future, is spatially heterogeneous, and areas that currently have large cattle populations are expected to be affected more severely than other regions.