Transition from octahedral to tetrahedral geometry causes the activation or inhibition by Zn<Superscript>2+</Superscript> of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> phosphorylcholine phosphatase

Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine, which is produced by the action of hemolytic phospholipase C on phosphatidylcholine or sphyngomielin, to generate choline and inorganic phosphate. Among divalent cations, its activity is dependent on Mg²⁺ or Zn²⁺. Mg²⁺ produced identical activation at pH 5.0 and 7.4, but Zn²⁺ was an activator at pH 5.0 and became an inhibitor at pH 7.4. At this higher pH, very low concentrations of Zn²⁺ inhibited enzymatic activity even in the presence of saturating Mg²⁺ concentrations. Considering experimental and theoretical physicochemical calculations performed by different authors, we conclude that at pH 5.0, Mg²⁺ and Zn²⁺ are hexacoordinated in an octahedral arrangement in the PchP active site. At pH 7.4, Mg²⁺ conserves the octahedral coordination maintaining enzymatic activity. The inhibition produced by Zn²⁺ at 7.4 is interpreted as a change from octahedral to tetrahedral coordination geometry which is produced by hydrolysis of the [ \textZn ²⁺ \textL ₂ ^{- 1} \textL ₂⁰ ( \textH ₂ \textO ) ₂ ] \left[ {{\text{Zn}}^{ 2+ } {\text{L}}_{ 2}^{ - 1} {\text{L}}_{ 2}^{0} \left( {{\text{H}}_{ 2} {\text{O}}} \right)_{ 2} } \right] complex.     

C-ansa-zirconocene complexes with O/S donor ligands: Novel homoleptic six coordinate 4-mercaptophenolate complex of Zr(IV)

New C-ansa-zirconocene complexes containing methoxythiophenolate and mercaptophenolate ligands have been synthesized and characterized. The reaction of (HSC₆H₄-n-OMe) (n = 2, 3 or 4) with [Zr{(t-Bu)HC(η⁵-C₅Me₄)(η⁵-C₅H₄)}Me₂] (1) led to the formation of monosubstituted complexes [Zr{(t-Bu)HC(η⁵-C₅Me₄)(η⁵-C₅H₄)}Me(κ,S-SC₆H₄-n-OMe)] (n = 2 (2); n = 3 (3)) and the disubstituted complex [Zr{(t-Bu)HC(η⁵-C₅Me₄)(η⁵-C₅H₄)}(κ,S-SC₆H₄-4-OMe)₂] (4). The complexes [Zr{(R)HC(η⁵-C₅Me₄)(η⁵-C₅H₄)}(κ,O-OC₆H₄-4-SH)₂] (R = t-Bu (6); R = CH₂CHCH₂ (7)) and [Zr(η⁵-C₅H₄)₂(OC₆H₄-n-SH)₂] (n = 3 (9); n = 4 (10)) have been synthesized using the corresponding dimethyl zirconocene and mercaptophenol. However, the reaction of [Zr{(t-Bu)HC(η⁵-C₅Me₄)(η⁵-C₅H₄)}Cl₂] (11) with 4-mercaptophenol in the presence of NEt₃ led to the formation of the first example of a homoleptic six-coordinate mercaptophenolate complex of zirconium, namely [HNEt₃]₂[Zr(κ,O-OC₆H₄-4-SH)₆] (12). Complex 12 can be obtained in higher yield by the reaction of ZrCl₄ with six equivalents of 4-mercaptophenol and NEt_3. The reaction of 12 with [Zr(η⁵-C₅H₄)₂Cl₂] gave the unexpected disubstituted complex [Zr(η⁵-C₅H₄)₂(OC₆H₄-4-SH)₂] (10). The molecular structures of 4 and 12 have been determined by single-crystal X-ray diffraction studies.     

Opposite fate of endocytosed CCR7 and its ligands: recycling versus degradation

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 play a crucial role for the homing of lymphocytes and dendritic cells to secondary lymphoid tissues. Nevertheless, how CCR7 senses the gradient of chemokines and how migration is terminated are poorly understood. In this study, we demonstrate that CCR7(-GFP) is endocytosed into early endosomes containing transferrin receptor upon CCL19 binding, but less upon CCL21 triggering. Internalization of CCR7 was independent of lipid rafts but relied on dynamin and Eps15 and was inhibited by hypertonic sucrose, suggesting clathrin-dependent endocytosis. After chemokine removal, internalized CCR7 recycled back to the plasma membrane and was able to mediate migration again. In contrast, internalized CCL19 was sorted to lysosomes for degradation, showing opposite fate for endocytosed CCR7 and its ligand.     

Probiotic properties of vaginal lactic acid bacteria to prevent metritis in cattle

AIMS: The isolation of bovine vaginal lactic acid bacteria (LAB) and the screening of their beneficial properties to select those that could be used as probiotics in the prevention of bovine metritis were performed. METHODS AND RESULTS: Out of 76 Lactobacillus sp. and seven Streptococcus sp. strains, a small number showed high- and medium hydrophobicity when the microbial adhesion to hydrocarbons method (MATH) was applied. In the agar plate diffusion test, a large number of strains inhibited vaginal bovine Escherichia coli 99/14 and human E. coli. This inhibition was due to acid. Only a few strains inhibited Actinomyces pyogenes 96/393, a pathogen isolated from bovine metritis. This inhibition remained after neutralization. The taxonomic identification of the selected strains was carried out by an amplified ribosomal DNA restriction analysis (ARDRA). Most of the strains were identified as Lactobacillus fermentum, a few as Lactobacillus gasseri and one as Lactobacillus rhamnosus. CONCLUSIONS: Bovine vaginal lactobacilli strains have differential surface properties. The strains selected are capable of inhibiting specific metritis pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results can be applied for future studies to design a probiotic product to prevent metritis in dairy postpartum cows.     

Critical Active-Site Residues Identified by Site-Directed Mutagenesis in Pseudomonas aeruginosa Phosphorylcholine Phosphatase, A New Member of the Haloacid Dehalogenases Hydrolase Superfamily

Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP), the product of the PA5292 gene, is synthesized when the bacteria are grown with choline, betaine, dimethylglycine, or carnitine. In the presence of Mg²⁺, PChP catalyzes the hydrolysis of both phosphorylcholine (PCh) and p-nitrophenylphosphate (p-NPP). PCh saturation curve analysis of the enzyme with or without the signal peptide indicated that the peptide was the fundamental factor responsible for decreasing the affinity of the second site of PChP for PCh, either at pH 5.0 or pH 7.4. PChP contained three conserved motifs characteristic of the haloacid dehalogenases superfamily. In the PChP without the signal peptide, motifs I, II, and III correspond to the residues ³¹DMDNT³⁵, ¹⁶⁶SAA¹⁶⁸, and K²⁴²/²⁶¹GDTPDSD²⁶⁷, respectively. To determine the catalytic importance of the D31, D33, T35, S166, K242, D262, D265, and D267 on the enzyme activity, site-directed mutagenesis was performed. D31, D33, D262, and D267 were identified as the more important residues for catalysis. D265 and D267 may be involved in the stabilization of motif III, or might contribute to substrate specificity. The substitution of T35 by S35 resulted in an enzyme with a low PChP activity, but conserves the catalytic sites involved in the hydrolysis of PCh (K_m1 0.03 mM, K_m2 0.5 mM) or p-NPP (K_m 2.1 mM). Mutating either S166 or K242 revealed that these residues are also important to catalyze the hydrolysis of both substrates. The substitution of lysine by arginine or by glutamine revealed the importance of the positive charged group, either from the amino or guanidinium groups, because K242Q was inactive, whereas K242R was a functional enzyme.     

Continuous enzymatic transesterification of sesame oil and a fully hydrogenated fat: effects of reaction conditions on product characteristics

An immobilized lipase from Thermomyces lanuginosus (TL IM) was employed to mediate the continuous transesterification of sesame oil and fully hydrogenated soybean oil (FHSBO) in a packed-bed reactor operating at 70 degrees C. Reactions between sesame oil (rich in LLL (15.97%), LOL (31.56%), and OLO (21.15%) [L = linoleic; O = oleic]) and the fully hydrogenated fat ((73.7% SSS, 26.3% SPS) [S = stearic; P = palmitic]) produced semi-solid fats. These products are complex mixtures of triacylglycerol (TAG) species whose compositions depend on reaction conditions. The dependence of the steady state product TAG profile on space time was determined for four initial weight ratios of sesame oil to hydrogenated fat (90:10, 80:20, 70:30, and 60:40). Except for the trial involving a weight ratio of sesame oil to FHSBO of 60:40, near equilibrium conditions were achieved at space times of 30 min-1 h. The chemical, physical, and functional properties of the product semi-solid fats were characterized. The predominant TAG species in the quasi-equilibrium products obtained from the mixture initially containing 90% (w/w) sesame oil and 10% FHSBO were LOL (26.22%) and OLO (21.92%). For transesterification of 80% sesame oil and 20% FHSBO, the major product species were OOP (21.27%), LOL (17.46%), and OLO (13.93%). OOP (24.38%) was the major product for reaction of 70% sesame oil with 30% FHSBO. Appropriate choices of reaction conditions and initial ratios of sesame oil to FHSBO lead to TAG with melting profiles and solid fat contents (SFC) similar to those of a variety of commercial products.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Effect of Mg, Si, and Sr on growth and antioxidant activity of the marine microalga Tetraselmis suecica

Efforts to increase the productivity of microalgal cultures have been focused on the improvement of photobioreactors, but little attention has been paid to the nutritional requirements of microalgae in order to improve culture media formulation. In this study, the main goal was obtaining a high productivity for Tetraselmis suecica (Chlorophyta) in semicontinuous culture by adding magnesium (Mg), silicon (Si), and strontium (Sr) at concentrations from 0.01 to 10 mM; at the time, the effect on steady-state cell density, biochemical composition, and antioxidant activity of T. suecica was evaluated. Because productivity is higher in high-density cultures, the work was focused many times to cell density. Mg (3 mM) and Sr (0.1 mM) added separately reached the highest steady-state cell density (7.0?×?10⁶?±?0.4 cells mL^?1) in comparison to control (4.2?±?0.1 cells mL^?1), but simultaneous addition had a synergic effect, achieving 8.7?×?10⁶?±?0.6 cells mL^?1. Silicon (3 mM) significantly affected the steady-state cell density, reaching 6.0?±?0.3 cells mL^?1 and increased the cell ash-free dry weight, reaching 127?±?7.9 pg cell^?1 in comparison to control (102.7?±?5.0 pg cell^?1), resulting in an ash-free dry weight productivity of 0.75?±?0.07 g?L^?1 day^?1. The highest fatty acids content and antioxidant activity, measured by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method were obtained with Sr 10 mM. Sr treatments showed a high correlation (R ²?=?0.98) between DPPH inhibition and polyphenolic content, explaining its high antioxidant activity. Therefore, the addition of Mg, Si, and Sr to culture medium of T. suecica is recommended to achieve high steady-state cell density in semicontinuous cultures.