The Streptococcus pyogenes NAD+ glycohydrolase modulates epithelial cell PARylation and HMGB1 release

Streptococcus pyogenes uses the cytolysin streptolysin O (SLO) to translocate an enzyme, the S. pyogenes NAD⁺ glycohydrolase (SPN), into the host cell cytosol. However, the function of SPN in this compartment is not known. As a complication, many S. pyogenes strains express a SPN variant lacking NAD⁺ glycohydrolase (NADase) activity. Here, we show that SPN modifies several SLO‐ and NAD⁺‐dependent host cell responses in patterns that correlate with NADase activity. SLO pore formation results in hyperactivation of the cellular enzyme poly‐ADP‐ribose polymerase‐1 (PARP‐1) and production of polymers of poly‐ADP‐ribose (PAR). However, while SPN NADase activity moderates PARP‐1 activation and blocks accumulation of PAR, these processes continued unabated in the presence of NADase‐inactive SPN. Temporal analyses revealed that while PAR production is initially independent of NADase activity, PAR rapidly disappears in the presence of NADase‐active SPN, host cell ATP is depleted and the pro‐inflammatory mediator high‐mobility group box‐1 (HMGB1) protein is released from the nucleus by a PARP‐1‐dependent mechanism. In contrast, HMGB1 is not released in response to NADase‐inactive SPN and instead the cells release elevated levels of interleukin‐8 and tumour necrosis factor‐α. Thus, SPN and SLO combine to induce cellular responses subsequently influenced by the presence or absence of NADase activity.     

Inhibition of haemolysis by the macrophage synthesized antioxidant, 7,8-dihydroneopterin

Abstract

Neopterin and the reduced form, 7,8-dihydroneopterin (78NP) are pteridines released from macrophages when stimulated with interferon-γ (IFN-γ) in vivo.^1,2 The role of 78NP in inflammatory response is unknown, though neopterin has been used clinically as a marker of immune cell activation due to its very fluorescent nature. 78NP is a potent antioxidant in a number of in vitro systems,^3–5 leading to the suggestion that it has a role in protecting macrophages from free radical damage during inflammation.³     

One‐Pot Solvent Free Synthesis and DNA Binding Studies of Thieno[2,3‐b]‐1,8‐Naphthyridines

Abstract

With the aim of evaluating interaction between double‐stranded calf thymus (ds)DNA and sulphur containing fused planar rings, the derivatives of 1,8‐naphthyridine containing thiono groups were synthesized by the condensation of 2‐mercapto‐3‐formyl[1,8]naphthyridines using 1‐chloroacetone, 2‐chloroacetamide, chloroaceticacid, and 2‐chloro‐1‐phenylethanone in the presence of anhydrous potassium carbonate as s catalyst under solvent free microwave irradiation. The structures of the compounds were elucidated on the basis of elemental analysis, IR, ¹H NMR, and mass spectra. The interaction of thieno[2,3‐b]‐1,8‐naphthyridine‐2‐carboxylic acid (TNC) (3a) with ct‐DNA was studied by UV‐Vis spectrophotometry, viscosity, thermal denaturation, as well as cyclic voltammetry experiments. On binding to DNA, the absorption spectrum underwent bathochromic and hypochromic shifts. Binding parameters, determined from spectrophotometric measurements indicated a binding constant of K _b =2.1×10⁶ M^?1. The thieno[2,3‐b]‐1,8‐naphthyridine‐2‐carboxylic acid (3a) increases the viscosity of sonicated rod‐like DNA fragments. The binding of TNC to DNA increased the melting temperature by about 4°C. The decrease in peak current heights and shifts of peak potential values are observed by the addition of calf thymus DNA in cyclic voltammetry studies.     

Combining a bio-based polymer and a natural antifoulant into an eco-friendly antifouling coating

Abstract

Biodegradable polymers are promising binders and carriers for natural antifoulants. In the present study, an antifouling (AF) coating was developed by adding a non-toxic AF compound (butenolide) to a bio-based and biodegradable poly(lactic acid)-based polyurethane. Mass loss measurement showed that the polymer degraded in seawater at a rate of 0.013?mg cm^?2?day^?1. Measurements showed that butenolide was released from the coatings into seawater over a period of at least three months. Both the concentration of butenolide in the coatings and the ambient temperature determined the release rate of butenolide. The results further demonstrate that incorporating rosin into the coatings increase the self-renewal rate of the polymer and facilitated the long-term release of butenolide from the coating. The results show that poly(lactic acid)-based polyurethane is a suitable polymer for butenolide-based AF coatings.     

Over‐expression of the molecular chaperone Hsp104 in Saccharomyces cerevisiae results in the malpartition of [PSI+] propagons

The ability of a yeast cell to propagate [PSI⁺], the prion form of the Sup35 protein, is dependent on the molecular chaperone Hsp104. Inhibition of Hsp104 function in yeast cells leads to a failure to generate new propagons, the molecular entities necessary for [PSI⁺] propagation in dividing cells and they get diluted out as cells multiply. Over‐expression of Hsp104 also leads to [PSI⁺] prion loss and this has been assumed to arise from the complete disaggregation of the Sup35 prion polymers. However, in conditions of Hsp104 over‐expression in [PSI⁺] cells we find no release of monomers from Sup35 polymers, no monomerization of aggregated Sup35 which is not accounted for by the proportion of prion‐free [psi^‐] cells present, no change in the molecular weight of Sup35‐containing SDS‐resistant polymers and no significant decrease in average propagon numbers in the population as a whole. Furthermore, they show that over‐expression of Hsp104 does not interfere with the incorporation of newly synthesised Sup35 into polymers, nor with the multiplication of propagons following their depletion in numbers while growing in the presence of guanidine hydrochloride. Rather, they present evidence that over‐expression of Hsp104 causes malpartition of [PSI⁺] propagons between mother and daughter cells in a sub‐population of cells during cell division thereby generating prion‐free [psi^?] cells.     

Bi‐modal sensitivity to monochromatic light by the mud snail Ilyanassa obsoleta †

Under laboratory conditions the mud snail Ilyanassa obsoleta exhibits a pronounced positive behavioral response to monochromatic light. The response spectrum for‐this positive phototaxis has maxima at 480 nm and 560–580 nm. The threshold intensities for this response are 2.06 x 10^‐7 /μW/cm² for 480 nm and 2.18 × 10^‐7 μW/cm² for 580 nm. These results are suggestive of Ilyanassa's possessing two visual pigments with different absorption maxima.     

Preparative Isolation and Purification of Hydroxyanthraquinones from Rheum tanguticum Maxim. on Normal Phase Silica Gel: Using a Flash Master Personal System

Abstract

A flash chromatography method for preparative separation and purification of five hydroxyanthraquinones from the Chinese medicinal herb Rheum tanguticum Maxim. ex Balf. was developed by using Flash Master Personal⁺ systems. The purities of the products, chrysophanol, physcion, emodin, aloe‐emodin, and rhein were all over 90%, determined by high performance liquid chromatography (HPLC). Their structures were ascertained by EI‐MS, ¹H‐ and ¹³C‐NMR data, and by co‐TLC and HPLC with the authentic samples available in our laboratory.     

Investigation of a sugar N‐formyltransferase from the plant pathogen Pantoea ananatis

Pantoea ananatis is a Gram‐negative bacterium first recognized in 1928 as the causative agent of pineapple rot in the Philippines. Since then various strains of the organism have been implicated in the devastation of agriculturally important crops. Some strains, however, have been shown to function as non‐pathogenic plant growth promoting organisms. To date, the factors that determine pathogenicity or lack thereof between the various strains are not well understood. All P. ananatis strains contain lipopolysaccharides, which differ with respect to the identities of their associated sugars. Given our research interest on the presence of the unusual sugar, 4‐formamido‐4,6‐dideoxy‐d ‐glucose, found on the lipopolysaccharides of Campylobacter jejuni and Francisella tularensis, we were curious as to whether other bacteria have the appropriate biosynthetic machinery to produce these unique carbohydrates. Four enzymes are typically required for their biosynthesis: a thymidylyltransferase, a 4,6‐dehydratase, an aminotransferase, and an N‐formyltransferase. Here, we report that the gene SAMN03097714_1080 from the P. ananatis strain NFR11 does, indeed, encode for an N‐formyltransferase, hereafter referred to as PA1080c. Our kinetic analysis demonstrates that PA1080c displays classical Michaelis–Menten kinetics with dTDP‐4‐amino‐4,6‐dideoxy‐d ‐glucose as the substrate and N¹⁰‐formyltetrahydrofolate as the carbon source. In addition, the X‐ray structure of PA1080c, determined to 1.7 Å resolution, shows that the enzyme adopts the molecular architecture observed for other sugar N‐formyltransferases. Analysis of the P. ananatis NFR11 genome suggests that the three other enzymes necessary for N‐formylated sugar biosynthesis are also present. Intriguingly, those strains of P. ananatis that are non‐pathogenic apparently do not contain these genes.     

Haltbarkeit von Silagen und die sie beeinflussenden Faktoren

Content of p‐coumaric (PCA) and ferulic (FA) acid was determined by the HPLC method in fourteen forbs with a potential utilization as forages (range of nutrient content per kg DM: 100 to 244gCP, 339 to 528 g NDF and 180–369 g ADF. PCA and FA were determined after methanol extraction in four fractions: free phenolic acids extracted into ether, ester‐bound phenolic acids after alkaline hydrolysis, glycoside‐bound phenolic acids after acid hydrolysis, and cell wall‐bound phenolic acids after alkaline hydrolysis of the solid residue after the extraction with methanol.

Cell wall‐bound phenols were quantitatively the most important fraction (50% of total PCA and 47% of total FA, respectively). The differences among plant species in total PCA plus FA content were significant (F‐value 775, P < 0.01). The range of total phenol content was 31.3 to 416.3 mg/100g DM, the overall mean was 84 mg/100g DM.

Content of phenolic acids was correlated neither with ADF, NDF or ADL content (R ² = 1–3%, P > 0.05) nor with CP degradability (R ² = 3% and R ² = 1% for PCA and FA, respectively, P > 0.05).

95.4% and 30.9% of total PCA, and 98.3% and 72.5% of total FA disappeared in the rumen from the sample of Glechoma hederacea (species with the highest phenol content) and from the sample of Galium aparine (species with low phenol content), respectively, within the four hour incubation interval.

It is presumed that in comparison with grasses, PCA and FA concentration in tested forbs represents a much lower risk in potential ruminant nutrition.     

Inhibitory effect of phenolic compounds and marine bacteria on larval settlement of the barnacle Balanus amphitrite amphitrite darwin

This study examined the inhibitory effect of 3 phenolic compounds and 12 strains of marine bacteria on the larval settlement of Balanus amphitrite amphitrite. The phenolic compounds used were phlorotannins, phloroglucinol and tannic acid. Phlorotannins are polymers of phloroglucinol (1,3,5‐trihydroxybenzene) known only from brown algae. Tannic acid, which exists in terrestrial plants, is composed of oligomers of phloroglucinol attached to a sugar molecule. The bacterial strains used were isolated from a natural biofilm. The following were investigated: 1) the toxicity of the phenolic compounds to B. a. amphirite in three different larval stages, viz. nauplius II, nauplius V and cyprid; 2) the potency of the compounds as inhibitors of larval settlement and the possible mechanism involved in settlement inhibition; and 3) the effects of the bacteria on larval settlement. The level of toxicity of the phenolic compounds varied widely for the larvae. Phlorotannins were most toxic, having LC₅₀ values ranging from 9.47 to 40.35 μg ml^‐1; phloroglucinol was least toxic, having LC₅₀ values of 235.12 to 368.28 μg ml^‐1. In general, nauplii were more sensitive to the toxicity of the phenolic compounds than cyprids. The greater sensitivity of nauplii may be due to their active feeding behavior, which exposes the interior of their bodies to the compounds by active intake. Phloroglucinol was the most potent settlement inhibitor, having an EC₅₀ value of 0.02 μg ml^‐1. Phlorotannins and tannic acid had EC₅₀ values of 1.90 μg ml^‐1 and 14.05 μg ml^‐1, respectively. Phloroglucinol appeared to inhibit larval settlement through a relatively non‐toxic mechanism as its LC₅₀ value was four orders of magnitude higher than its EC₅₀ value. The high potency of phloroglucinol indicates that a simple constituent of a complex natural compound can be more effective than the natural compound itself. Larval settlement bioassays with monospecies bacterial films indicated that some of the bacterial species were inhibitory to larval settlement while the others showed no effect. None of the bacterial strains in this study induced larval settlement.     

Interactive effects of chromate and arsenate on their uptake and speciation in Pteris ensiformis

Background and aims

Arsenate (AsV) and chromate (CrVI) inhibit each other’s uptake and translocation in As-hyperaccumulator Pteris vittata. In the present study, we extended the research to As-sensitive plant Pteris ensiformis to better understand the mechanism of their interactions.

Methods

Plants were exposed to 0, 0.75 or 7.5 mg L^?1 AsV and 0, 0.52, or 5.2 mg L^?1 CrVI for 7 d in hydroponics. Arsenic and Cr speciation were determined in nutrient solutions and plant biomass.

Results

P. ensiformis accumulated high levels of As and Cr in the rhizomes and roots with low levels in the fronds. However, P. ensiformis was much more effective in taking up Cr than As, as much more Cr was accumulated in the roots (306–6015 vs. 87–642 mg kg^?1). AsV and CrVI increased each other’s uptake in the rhizomes and roots when co-present. The AsV and CrVI taken up by P. ensiformis were reduced to arsenite (AsIII) and chromite (CrIII), possibly serving as detoxification mechanism.

Conclusions

Uptake of As and Cr induced oxidative stress as indicated by increased lipid peroxidation and electrical conductivity. Arsenic and Cr increased each other’s uptake by P. ensiformis.

     

Homo-C-Nucleoside Analogs† II. Synthesis and Anomeric Configuration of 4-(2,5-Anhydro-D-Gluco-PEntitol-1-YL)-2-Phenyl2H-1,2,3-Triazole††

Abstract

Treatment of 4-(D-gluco-pentitol-l-y1)-2-pheny1–2H-1,2,3-triazole (1) with p-toluenesulfonyl chloride in pyridine solution, afforded the homo-C-nucleoside analog, 4-(2,5-anhydro-D-gluco-pentitol-1-yl)-2-phenyl-2H-1,2,3-triazole (2) as well as its partial p-toluenesulfonyl derivative (3). 4-(5-Chloro-5-deoxy-D-gluco-pentitol-1-yl)-2-phenyl-2H-1,2,3-triazole (8), was isolated as a byproduct from the reaction. The structure and anomeric configuration of 2 was determined by acylation, ¹H, ¹³C NMR, and NOE, spectroscopy as well as mass spectrometry.

     

Structures and genetics of Kdo-containing O-antigens of Cronobacter sakazakii G2706 and G2704, the reference strains of serotypes O5 and O6

Cronobacter sakazakii G2706 and G2704 are the reference strains of serotypes O5 and O6 in the serological classification of this species proposed recently. Mild acid degradation of the lipopolysaccharides of both strains resulted in cleavage of the O-polysaccharide chains at the acid-labile linkage of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to yield oligosaccharides representing repeating units of the O-polysaccharides. The oligosaccharides and alkali-degraded lipopolysaccharides were studied by sugar analysis along with 1D and 2D ¹H and ¹³C NMR spectroscopy, and the following O-polysaccharide structures were established:The structure of strain G2706 is unique among the known bacterial polysaccharide structures, whereas that of strain G2704 is identical to the structure of Cronobacter malonaticus 3267 [MacLean, L. L.; Vinogradov, E.; Pagotto, F.; Farber, J. M.; Perry, M. B. Biochem. Cell Biol.2009, 87, 927–932], except for that the latter lacks O-acetylation. Putative functions of the genes in the O-antigen gene clusters of C. sakazakii strains studied are in agreement with the O-polysaccharide structures.     

Modelling of Structural and Vibrational Properties of Poly(p-Phenylene) and Polypyrrole Using Molecular Orbital Methods

Abstract

This paper concentrates on two very important conducting polymers poly(p-phenylene) and polypyrrole. Detailed atomistic molecular models have been developed with the help of ab initio and semi-empirical quantum mechanical calculations using the Cerius² and WinMOPAC (version 6.0) programs.

Their optimised geometry had been calculated and compared with experimental X-ray diffraction data. The simulated and experimental vibrational spectra of biphenyl as well as isolated pyrrole monomers and oligomers from n = 1 and 2, where n is the number of structural repeat units used, have been computed using the ab initio 3–21G basis set. The results obtained are compared with experimental data for the case of biphenyl and for oligomers with n = 2 to 5 for both neutral benzenoid and quinonoid oligopyrroles, from semi-empirical predictions obtained by AM1 and PM3. The trends in the computed harmonic force fields, vibrational frequencies and intensities are monitored as a function of the chain length. The data are analyzed in conjunction with the trends in computed equilibrium geometries.     

Artificial insemination in cattle with DNA‐treated sperm

Abstract

We have investigated the use of sperm cells as vectors for transferring exogenous DNA into the genome of cattle by artificial insemination with DNA‐treated sperm. First we demonstrated the DNA‐binding ability of cattle sperm with radioactively labeled DNA. For artificial insemination ejaculated semen was washed and incubated with 1 μg DNA/10⁶ sperm for one hour at 37°C. Three hundred synchronized heifers were inseminated once with a dose of 40×10⁶ sperm. Forty‐five calves and 41 fetuses were obtained. Southern analysis revealed in one calf a signal after probing with the 1 kb Pst I fragment of pSV2‐cat.     

Nitrogen‐isotope fractionation in the nitrate respiration by the marine bacterium Serratia marinorubra

Abstract

The isotopic composition (δ¹⁵N) of nitrite produced during nitrate respiration by both growing and washed cells of the marine bacterium, Serratia marinorubra, was determined. In both the growing and washed cells, δ¹⁵N of the nitrite changed considerably with time. At least two reaction steps, producing different isotope effects (active transport of nitrate across membranes and reduction of nitrate to nitrite), appear to be involved. With washed cells, a fractionation factor as high as 1.039 was obtained—the highest ever reported for biologicalnitrate reduction. The physiological state of nitrate‐respiring bacteria in oxygen‐depleted subsurface waters of the sea is discussed from the viewpoint of isotope fractionation.     

Volatile substances from adult extracts induce larval settlement of the barnacle balanus amphitrite *

The volatile substance extracted from conspecific adults induces larval settlement of the barnacle Balanus amphitrite. The settlement inducing activity of the volatile fractions was checked monthly from June, 1997 to December, 1998. Both water soluble extracts and volatile fractions from the barnacle were prepared by the steam distillation. The active cue in the volatile fraction was always extracted with n‐pentane under acidic conditions, although settlement inducing activity varied with the sample. GC‐MS analysis of the active and inactive pentane fractions revealed 1, 2, 3‐trimethyl‐benzene as the settlement inducing substance. Commercially available 1, 2, 3‐trimethylbenzene also showed high settlement inducing activity at a concentration of 0.8 × 10^‐12 M (100 pg 1^‐1). This substance was detected at concentrations of more than 7 ng g^‐1 of wet barnacle (equivalent to 0.6 × 10^‐12M, equal to 70pg1^‐1) by GC analysis. These results indicate that 1,2,3‐trimethylbenzene in the volatile fractions acts as a chemical cue for larval settlement. Monthly variation in the settlement inducing activity was observed, which synchronized with the breeding season of the barnacle. This observation suggests that the barnacle produced the chemical cue in the gonad during maturation or accumulated it from the environment.     

News notes

FERTILITY AND SURVIVAL, POPULATION PROBLEMS FROM MALTHUS TO MAO TSE‐TUNG: Alfred Sauvy. Translated by Christine Brooke‐Rose, Criterion Books Inc., New York, 1961, 232 pp. (simultaneously Chatto &; Windus Ltd., London).

^l COLOMBO, BERNARDO, 1961. In: Rivista Internazionale di Science Sociali, 64: (5): pp. 505–12.

MIRAGE OF HEALTH: Rene Dubos. Dou‐bleday and Company, Inc. (Anchor Books), New York, 1961, 230 pp., $0.95.

POPULATION CONTROL, THE IMMINENT WORLD CRISIS: Melvin G. Shimm and Robinson O. Everett (Ed.). Oceana Publications, Inc., New York, 1961, pp. iv + 254, $6.00.     

Chromosomal Mapping of HSPCB and MYL1 Expressed Abundantly in the Bovine Fetus

Abstract

Chromosomal mapping of expressed sequence tags for HSPCB and MYL1 expressed abundantly in the bovine fetus was performed by analyzing bovine/murine somatic cell hybrid DNAs with polymerase chain reaction (PCR) using primers specific for those 3^′‐untranslated regions. HSPCB and MYL1 were assigned to bovine chromosomes 23 and 2, respectively.     

An Efficient Route to Novel 4,5‐Di‐ and 2,4,5‐Tri Substituted Imidazoles from Imidazo[1,5‐a]‐1,3,5‐triazine (5,8‐Diaza‐7,9‐dideazapurine) Derivatives

Two new types of imidazole derivatives: N‐(2‐R¹‐5‐R²‐1H‐imidazol‐4‐yl) thioureas 7a–g and N‐(2‐R¹‐5‐R²‐1H‐imidazol‐4‐yl) formamides 8b,c,g were obtained in high yields by the hydrolytic degradation of 6‐R¹‐8‐R²‐2‐thioxo‐2,3‐dihydroimidazo[1,5‐a]‐1,3,5‐triazin‐4(1H)‐ones 5a–g and 6‐R¹‐8‐R²‐imidazo[1,5‐a]‐1,3,5‐triazin‐4(3H)‐ones 6b,c,d, respectively. The tautomeric preferences of the new imidazoles were determined.