New pyrrolopyrimidin-6-yl benzenesulfonamides: potent A2B adenosine receptor antagonists

A new series of 4-(1,3-dialkyl-2,4-dioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl)benzenesulfonamides has been identified as potent A2B adenosine receptor antagonists. The products have been evaluated for their binding affinities for the human A2B, A1 and A3 adenosine receptors. 6-(4-{[4-(4-Bromobenzyl)piperazin-1-yl]sulfonyl}phenyl)-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione (16) showed a high affinity for the A2B adenosine receptor (IC50=1 nM) and selectivity (A1: 183x; A3: 12660x). Synthesis and SAR of this novel class of compounds showing improved absorption properties is presented herein.     

Recycling Endosome Membrane Incorporation into the Leading Edge Regulates Lamellipodia Formation and Macrophage Migration

In comparison to our knowledge of the recycling of adhesion receptors and actin assembly, exactly how the cell controls its surface membrane to form a lamellipodium during migration is poorly understood. Here, we show the recycling endosome membrane is incorporated into the leading edge of a migrating cell to expand lamellipodia membrane. We have identified the SNARE complex that is necessary for fusion of the recycling endosome with the cell surface, as consisting of the R‐SNARE VAMP3 on the recycling endosome partnering with the surface Q‐SNARE Stx4/SNAP23, which was found to translocate and accumulate on the leading edge of migrating cells. Increasing VAMP3‐mediated fusion of the recycling endosome with the surface increased membrane ruffling, while inhibition of VAMP3‐mediated fusion showed that incorporation of the recycling endosome is necessary for efficient lamellipodia formation. At the same time, insertion of this recycling endosome membrane also delivers its cargo integrin α5β1 to the cell surface. The loss of this extra membrane for lamellipodia expansion and delivery of cargo in cells resulted in macrophages with a diminished capacity to effectively migrate. Thus, the recycling endosome membrane is incorporated into the leading edge and this aids expansion of the lamellipodia and simultaneously delivers integrins necessary for efficient cell migration.     

Aging-related oxidative stress depends on dietary lipid source in rat postmitotic tissues

We investigate mitochondrial-lipid peroxidation of mitotic (liver) and postmitotic (heart and skeletal muscle) tissues of rats fed lifelong on two different lipid sources: virgin olive oil (monounsaturated fatty acids) and sunflower oil (n–6 polyunsaturated fatty acids). Two groups of 80 rats each were fed over 24 months on a diet differing in the lipid source (virgin olive oil or sunflower oil). Twenty rats per group were killed at 6, 12, 18, and 24 months; liver, heart, and skeletal muscle mitochondria were isolated and the lipid profile, hydroperoxides, vitamin E, and ubiquinone as well as catalase activity measured. Lipid peroxidation was higher in postmitotic tissues, and sunflower oil led to a higher degree of polyunsaturation and peroxidation. The levels of -tocopherol adapted to oxidative stress and preferentially accumulated during aging in heart and skeletal muscle. In conclusion, the type of dietary fat should be considered in studies on aging, since oxidative stress is directly modulated by this factor. This study confirms that postmitotic tissues are more prone to oxidative stress during aging and proposes a hypothesis to explain this phenomenon.     

Environmental factors affecting tissue regeneration of the reef--building coral Montastraea annularis (Faviidae) at Los Roques National Park,Venezuela

In this study, the rates of tissue regeneration and recovery from injuries that emulated the bites of either butterfly or parrotfish on colonies of Montastraea annularis exposed to different sedimentation regimesp were determined. Two small reef patches were chosen close to key Dos Mosquises, north of the Venezuclan mainland. Sixteen colonies (8 treatments + a single replicate) were artificially damaged at each patch and their recovery was monitored for three months by photographic means. The lesions were inflicted using two different techniques: scratching the polyps with a hard-nylon brush to resemble parrotfish (Scaridae) damages (Lesions Type 1) or jetting out the tissue with a syringe to simulate butterflyfish (Chaetondontidae) bites (Lesions Type 2). The diameter of the wounds ranged from 5 (small lesion) to 8 cm (large lesions) and both kinds were inflicted on the top and bottom of the colonies, with a single replicate for each treatment. The main factors affecting the recovery of the colonies' surface were lesion features (type, position and size), turbidity and chiefly, the sedimentation rate. While lesion recovery was slow where sedimentation and resuspension rates were high, tissue regeneration was improved under low sedimentation and resuspension conditions. Moreover, lesions located at the bottom of colonies regenerated completely, whereas sediments frequently covered top lesions and limited their recovery. More than 60% of the colonies with small lesions recovered almost completely in less than 90 days, whereas those with larger injuries frequently showed extensions of their damage and increased mortality. Tissue-only lesions (LT2) regenerated two to three times faster than those involving both tissue and skeletal damage (LT1). Other variables not controlled in this study, such as diseases, encrusting organisms overgrowth and herbivory introduced further variability to the regeneration rates.     

Structure of the β-Galactosidase Gene from Thermus sp. Strain T2: Expression in Escherichia coli and Purification in a Single Step of an Active Fusion Protein

The nucleotide sequence of both the bgaA gene, coding for a thermostable β-galactosidase of Thermus sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (M_r, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the bgaA gene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the β-galactosidase of Bacillus stearothermophilus allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric β-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70°C and a K_m value of 1.6 mM with o-nitrophenyl-β-d-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70°C.β-d-Galactosidase (EC 3.2.1.23) catalyzes the hydrolysis of β-1,4-d-galactosidic linkages. This enzyme is distributed in numerous microorganisms, plants, and animal tissues. The application of β-galactosidase to the hydrolysis of lactose in dairy products, such as milk and cheese whey, has received much attention (7, 21), and in this regard, thermostable β-galactosidases have attracted increasing interest because of their potential usefulness in the industrial processing of lactose-containing products (21). Thermostable enzymes have a number of generally recognized advantages in industrial applications, such as associated chemical resistance and reduced chances of microbial growth at high temperatures (15, 19). Nevertheless, relatively few studies have been conducted on β-galactosidases from thermotolerant or thermophilic bacteria, and as far as we know, only four genes encoding these enzymes have been cloned (5, 10, 11, 13, 16, 18).An important property that has received little attention in the literature is the level of purity of commercial preparations of β-galactosidases, especially with regard to the presence of other enzymes, such as proteases. These contaminants could have a severe impact on the stability of the enzyme, leading to undesirable changes in dairy products during storage (21). To prevent these, a new method was developed to purify the β-galactosidase (LacZ) of Escherichia coli by fusing to its N terminus the choline-binding domain (ChBD) of the pneumococcal autolytic amidase LytA (23). This system allowed the purification of E. coli β-galactosidase in a single step by affinity chromatography on DEAE-cellulose (23). Thus, it appeared interesting to test whether this procedure could also be used in the purification of a thermostable enzyme in order to circumvent contamination problems.This paper reports the molecular characterization of the bgaA gene, encoding the β-galactosidase (BgaA) of Thermus sp. strain T2, and describes the construction of a ChBD-BgaA chimera which retains the biochemical properties of the native enzyme and can be purified in a single chromatographic step.     

Survival in Soil of Different Toluene-Degrading Pseudomonas Strains after Solvent Shock

We assayed the tolerance to solvents of three toluene-degrading Pseudomonas putida strains and Pseudomonas mendocina KR1 in liquid and soil systems. P. putida DOT-T1 tolerated concentrations of heptane, propylbenzene, octanol, and toluene of at least 10% (vol/vol), while P. putida F1 and EEZ15 grew well in the presence of 1% (vol/vol) propylbenzene or 10% (vol/vol) heptane, but not in the presence of similar concentrations of octanol or toluene. P. mendocina KR1 grew only in the presence of heptane. All three P. putida strains were able to become established in a fluvisol soil from the Granada, Spain, area, whereas P. mendocina KR1 did not survive in this soil. The tolerance to organic solvents of all three P. putida strains was therefore assayed in soil. The addition to soil of 10% (vol/wt) heptane or 10% (vol/wt) propylbenzene did not affect the survival of the three P. putida strains. However, the addition of 10% (vol/wt) toluene led to an immediate decrease of several log units in the number of CFU per gram of soil for all of the strains, although P. putida F1 and DOT-T1 subsequently recovered. This recovery was influenced by the humidity of the soil and the incubation temperature. P. putida DOT-T1 recovered from the shock faster than P. putida F1; this allowed the former strain to become established at higher densities in polluted sites into which both strains had been introduced.     

Population patterns of Mexican corn rootworm (Coleoptera: Chrysomelidae) adults indicated by different sampling methods

The Mexican corn rootworm, Diabrotica virgifera zeae Krysan & Smith, is a serious pest of corn, Zea mays L., in several areas of Texas. Recent demonstrations of areawide adult control suggest this tactic has promise for rootworm management, but additional information regarding treatment thresholds and sampling methodology is needed. In 2000 and 2001 we examined the influence of distance into the field on rootworm captures by CRW and Pherocon AM traps, the fidelity of trap captures to population estimates from visual counts of beetles on plants (whole plant samples), and the seasonal population patterns indicated by each sampling method. Only the CRW trap consistently indicated reduced trap captures at the field margin compared with other distances. However, trends for the AM trap and whole plant samples suggested sampling on the field margin should be avoided. Population estimates at other distances into the field (2-30 m) were usually statistically similar. Thus, monitoring does not require trap placement far into the field. Both trap types indicated population peaks after flowering in corn, whereas plant samples indicated peak populations during tasseling and flowering. Both the CRW trap and plant samples showed the proportion of female beetles increased as the season progressed, but the CRW trap underestimated the proportion of females until after flowering. Regressions relating captures by traps to counts from plant samples indicated efficiency of both traps increased with increasing plant development. Our findings should increase acceptance of the CRW trap by producers and consultants and provide a rationale for development of improved, plant growth stage-specific treatment thresholds.     

Effect of ammonia on the methanogenic activity of methylaminotrophic methane producing Archaea enriched biofilm

Ammonia is a metabolic product in the decomposition of protein wastes, and has a recognized inhibitory effect on methanogenesis; this effect has been slightly quantified on methanogenic biofilms and particularly those populated by methanogenic Archaea which produce ammonia as a catabolic product from methylated amines. This paper presents studies on the effect of ammonia on maximum methanogenic activity of anaerobic biofilms enriched by methylaminotrophic methane producing Archaea (mMPA). The effect of unionized free ammonia on the specific maximum methanogenic activity of a mMPA enriched biofilm was studied, using 250 mL flasks containing ceramic rings colonized by 30 day-old experimental biofilm and adding 48.8 (control system), 73.8, 98.8, 148.8, 248.8, 448.8 and 848.8 mg NH(3)-N/L. The systems were maintained for ten days at a pH of 7.5 and temperature of 37 degrees C. The results showed that at 848.8 mg NH(3)-N/L, biofilm methane production required 36 h adaptation period, prior to entering into maximum production phase. The highest maximum methanogenic activity reached a value of 2.337+/-0.213 g COD methane/g VSS *day when 48.8 mg NH(3)-N/L was added, and inhibition was clearly observed in those systems above 148.8 mg NH(3)-N/L, producing under 1.658+/-0.185 g COD methane/g VSS *day. The lowest methanogenic activity reached was 0.639+/-0.162 g COD methane/g VSS *day at the system added with 848.8 mg NH(3)-N/L. When applying the Luong and non-competitive inhibition models, the best fit was obtained with the non-competitive model, which predicted 50% inhibition of methanogenic activity at 365.288 mg NH(3)-N/L.     

Electron paramagnetic resonance study of the migratory ant Pachycondyla marginata abdomens

Electron paramagnetic resonance was used to investigate the magnetic material present in abdomens of Pachycondyla marginata ants. A g congruent with 4.3 resonance of high-spin ferric ions and a very narrow g congruent with 2 line are observed. Two principal resonance broad lines, one with g > 4.5 (LF) and the other in the region of g congruent with 2 (HF), were associated with the biomineralization process. The resonance field shift between these two lines, HF and LF, associated with magnetic nanoparticles indicates the presence of cluster structures containing on average three single units of magnetite-based nanoparticles. Analysis of the temperature dependence of the HF resonance linewidths supports the model picture of isolated magnetite nanostructures of approximately 13 nm in diameter with a magnetic energy of 544 K. These particles are shown to present a superparamagnetic behavior at room temperature. The use of these superparamagnetic particle properties for the magnetoreception process of the ants is suggested.