Initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa related to the human cytosolic 5'-nucleotidase I.

The 5'-nucleotidases constitute a ubiquitous family of enzymes that catalyze either the hydrolysis or the transfer of esterified phosphate at the 5' position of nucleoside monophosphates. These enzymes are responsible for the regulation of nucleotide and nucleoside levels in the cell and can interfere with the phosphorylation-dependent activation of nucleoside analogs used in therapies targeting solid tumors and viral infections. In the present study, we report the initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa that is related to the human cytosolic 5'-nucleotidase I. X. fastidiosa is a plant pathogenic bacterium that is responsible for numerous economically important crop diseases. Biochemical assays confirmed the phosphatase activity of the recombinant purified enzyme and revealed metal ion dependence for full enzyme activity. In addition, we investigated the involvement of Xf5'-Nt in the formation of X. fastidiosa biofilms, which are structures that occlude the xylem vessels of susceptible plants and are strictly associated with bacterial pathogenesis. Using polyclonal antibodies against Xf5'-Nt, we observed an overexpression of Xf5'-Nt during the initial phases of X. fastidiosa biofilm formation that was not observed during X. fastidiosa planktonic growth. Our results demonstrate that the de/phosphorylation network catalyzed by 5'-nucleotidases may play an important role in bacterial biofilm formation, thereby contributing novel insights into bacterial nucleotide metabolism and pathogenicity.

Enzymatic variability among Brazilian Pythium insidiosum isolates.

BACKGROUND: Pythium insidiosum is an oomycete classified in the kingdom Stramenopila. P. insidiosum hyphae are not able to initiate infection without the secretion of hydrolytic enzymes, which are considered an important factor in microbial virulence. AIMS: To evaluate the extracellular enzymatic activity of 14 Brazilian P. insidiosum isolates and a standard strain (ATCC 58637) by the API-ZYM System screening method. METHODS: Zoospores were grown in RPMI 1640 broth, and 65 µL of the liquid phase were inoculated in each cupule of the API-ZYM strips. RESULTS: Differences in the enzymatic activities were observed among the isolates, although phosphohydrolases and ester hydrolases were conspicuous among all isolates. ß-glucosidase was also present in most of the isolates. Enzymatic activities of α-glucosidase and chymotrypsin were not observed, differing from a previous study involving Australian isolates and intracellular enzymes. CONCLUSIONS: The discrepancy in the enzymatic profile observed among Brazilian P. insidiosum isolates reflects the phenotypic variations found in susceptibility tests.

Detection of endometrial intraepithelial neoplasia (EIN) in postmenopausal endometrium.

OBJECTIVES: To detect the presence of endometrial intraepithelial neoplasia (EIN) in the endometrium of postmenopausal patients. PATIENTS AND METHODS: Sixty-three postmenopausal patients with endometrial polyps (n=48), hyperplasia (n=12) and endometrioid carcinoma (n=3) were enrolled for this study. The diagnosis of EIN was made by using morphological criteria and immunohistochemical methods for detection of PTEN and bcl-2. RESULTS: EIN lesions were found in cases of endometrial polyp (n=1), atrophic endometrium (n=1) and in hyperplasia (n=1). The glands were packed, showed cytological atypia and were negative for both PTEN and bcl-2. Three patients with endometrial hyperplasia had isolated PTEN-negative glands but they were still bcl-2 positive. CONCLUSIONS: The use of immunohistochemical methods helps detect the presence of EIN in the postmenopausal endometrium but does not substitute the morphological criteria for this diagnosis.

The pH-induced glycosylation of secreted phosphatases is mediated in Aspergillus nidulans by the regulatory gene pacC-dependent pathway.

In this communication, we show that the pacC(c)14 mutation drastically reduced the mannose and N-acetylglycosamine content of the pacA-encoded acid phosphatase secreted by the fungus Aspergillus nidulans when grown at 22 degrees C, pH 5.0, compared to a control strain. The staining after PAGE was not observed for the pacA-encoded acid phosphatase, while the palD-encoded Pi-repressible alkaline phosphatase had an altered electrophoretic mobility. In addition, the secreted acid phosphatase also had a reduced number of isoforms visualized by staining after IEF and glycosylation had a protective effect against its heat inactivation. We also show that a full-length version of gene pacC-1 cloned from Neurospora crassa complemented the pacC(c)14 mutation of A. nidulans, including the remediation of both the acid and alkaline Pi-repressible phosphatases secreted at pH 5.0, which indicates that glycosylation of secreted phosphatases is mediated in A. nidulans by the conserved PacC pathway that governs pH-responsive gene expression.

A technique for mycelial development of ectomycorrhizal fungi on agar media.

A technique was established to study ectomycorrhizal fungi on agar media. Petri dishes, 60 mm in diameter, containing 10 mL of culture medium covered with a cellophane disk were used for easy collection of the mycelium after growth. For analysis of fungal biomass production, a sterilized cellophane sheet was placed on the medium's surface. Inoculation was achieved by placing a mycelial block onto the center of the cellophane sheet and then incubating at 25 degrees C in the dark. Colony radial growth was measured and biomass dry wt was determined. Fresh mycelia were homogenized with 10 mL of acetate buffer (pH 5.5) for enzyme analysis. A crude extract was obtained by adding all culture medium to 90 mL of distilled water and homogenizing in a Potter. Reducing sugars, enzyme concentration, and pH were determined. Three fungal strains, Suillus collinitus, Pisosithus arrhizus, and Hebeloma cylindrosporum, were grown in different culture media (potato dextrose agar or Pintro's medium). Parameters measured over time included glucose concentration, phosphatase activity, biomass, and pH.

Sucrose-phosphate phosphatase from Anabaena sp. strain PCC 7120: isolation of the protein and gene revealed significant structural differences from the higher-plant enzyme.

The present study describes the first isolation and characterization of a prokaryotic protein and gene for sucrose-phosphate phosphatase (SPP), the enzyme that catalyzes the terminal step in sucrose synthesis. For gene isolation, a 2,015-bp DNA fragment containing an open reading frame with about 31% amino acid identity to Synechocystis SPS was amplified from Anabaena sp. PCC 7120 DNA. Surprisingly, expression of the putative gene in Escherichia coli demonstrated that it encoded an SPP protein. The expressed protein cross-reacted with antibodies against the native form of Anabaena SPP and its biochemical properties were identical to those of the enzyme purified from the cyanobacterial cells. Comparisons of the Anabaena SPP with the higher-plant enzyme revealed important differences in the C-terminal region, molecular mass, subunit composition and immunoreactivity. Nevertheless, two conserved motifs, including four invariant aspartate residues similar to those found in members of the phosphohydrolase superfamily, were identified in the Anabaena SPP deduced amino acid sequence.

Kinetic properties of purified Pseudomonas aeruginosa phosphorylcholine phosphatase indicated that this enzyme may be utilized by the bacteria to colonize in different environments.

Pseudomonas aeruginosa phosphorylcholine phosphatase from periplasmic extracts of bacteria grown on choline as the sole carbon and nitrogen source was purified to homogeneity. The enzyme represented nearly 1% of the total protein found in the periplasmic space and is a monomer of approximately 53 kDa with an isoelectric point of 7.5. The optimum pH with phosphorylcholine was in the range of 5-8; with phosphorylethanolamine there was a peak at pH 6, and with p-nitrophenyl-phosphate (p-NPP) the optimum was at pH 5. Studies carried out at pH 5 indicated: i) For the three substrates above, Mg2+, Zn2+, or Cu2+ was necessary for maximal activity. ii) With p-NPP, these cations bound to the free enzyme in an ordered bireactant system. iii) With phosphorylethanolamine, Mg2+, Zn2+, or Cu2+ bound to the free enzyme in an at random bireactant system. iv) With phosphorylcholine, maximal activity was obtained with cation concentrations as low as 100 nM. v) Al3+ ions were inhibitors of the enzyme activity. The n (Hill coefficient) values for the inhibition by Al3+ with phosphorylcholine or p-NPP were 1 or 4, respectively. vi) The enzyme exhibited two affinity sites for phosphorylcholine. With Mg2+, a site with a Km value of 0.5 mM was detected; the corresponding Vmax was 25 micromol min-1 (mg protein)-1; without Mg2+, the enzyme displayed Km and Vmax values of 0.09 mM and 4.2 micromol min-1 (mg protein)-1, respectively. Studies carried out at pH 7.4 indicated: i) The enzyme could not catalyze the hydrolysis of p-NPP, and phosphorylethanolamine was a poor substrate in either the presence or absence of divalent cations. ii) The enzyme activity measured with phosphorylcholine was independent of divalent cations or was not inhibited by Al3+ ions. iii) With or without Mg2+, the enzyme exhibited two affinity sites for phosphorylcholine; the Km values were 0.05 mM and 0.5 mM with their corresponding Vmax of 5.6 and 25 micromol min-1 (mg protein)-1, respectively.