RESUMEN

Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate alpha-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins. S. nigra TRITC-conjugated lectin bound to this biofilm, while M. amurensis FITC-conjugated lectin did not. S. nigra TRITC-conjugated lectin binding was inhibited by incubation with alpha2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrix-assisted laser desorption ionization-time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were two- to fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing alpha2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.

Asunto(s)

RESUMEN

The feasibility of mesophilic anaerobic co-digestion of landfill leachate and sewage sludge was examined in a bench-scale experiment. Three complete-mix, flow-through digesters were operated in a semi-continuous mode. During both phases of research all digesters received 500 ml d(-1) of raw sludge and Reactor 1 was always the control reactor--fed sludge only. During Phase 1, leachate volumes less than 12% of the sludge volume were fed to Reactors 2 and 3. During Phase 2 larger amounts of leachate were added, exceeding 20% of sludge volume which led to an overall decrease in the hydraulic residence time of the digesters. All reactors achieved stable operation, which indicated that the co-digestion of sewage sludge and landfill leachate is feasible During Phase 1, an increase in the average daily methane production from 2.5 l d(-1) to 3.1 l d(-1) and 3.2 l d(-1) was observed; the biomethanation production (BMP) increased from 0.46 to 0.6 m3 - 0.7 m3 CH4 (kg VS rem.)(-1). The average volatile solids reduction (VSR) increased from 46.1% to 48.6% and 49.0%. In Phase 2, the total methane production in the control reactor was significantly higher, at 4.6 l d(-1), while the addition of larger, by volume, amounts of leachate, decreased the methane production to 4.3 l d(-1) and 4.2 l d(-1), respectively. The average BMP values were 0.8, 0.87, and 0.81 m3 CH4 (kg VS rem.)(-1), respectively. In Phase 2, leachate addition decreased the average VSR from 51% to 49% and 45.6%. After calculating that leachate addition to digesters would not increase heavy metal concentrations in the produced biosolids it was concluded that mesophilic anaerobic co-digestion of sewage sludge and landfill leachate is feasible, and provides a promising alternative to aerobic co-treatment.

Asunto(s)

RESUMEN

To facilitate studies of the molecular determinants of host-meningococcal lipooligosaccharide (endotoxin) interactions at patho-physiologically relevant endotoxin concentrations (i.e. < or =10 ng/ml), we have generated acetate auxotrophs NMBACE1 from encapsulated Neisseria meningitidis (serogroup B, strain NMB) and NMBACE2 from an isogenic bacterial mutant lacking the polysialic acid capsule. Growth of the auxotrophs in medium containing [(14)C]acetate yielded (14)C-lipooligosaccharides containing approximately 600 cpm/ng. Gel sieving resolved 14C-lipooligosaccharide-containing aggregates with an estimated molecular mass of > or =20 x 10(6) Da (peak A) and approximately 1 x 10(6) Da (peak B) from both strains. Lipooligosaccharides in peaks A and B had the same fatty acid composition and SDS-polyacrylamide gel electrophoresis profile. 14C-Labeled capsule copurified with (14)C-lipooligosaccharides in peak B from NMBACE1, whereas the other aggregates contained only 14C-lipooligosaccharide. For all aggregates, lipopolysaccharide-binding protein and soluble CD14-induced delivery of lipooligosaccharides to endothelial cells and cell activation correlated with disaggregation of lipooligosaccharides. These processes were inhibited by the presence of capsule but unaffected by the size of the aggregates. In contrast, endotoxin activation of cells containing membrane CD14 was unaffected by capsule but diminished when endotoxin was presented in larger aggregates. These findings demonstrate that the physical presentation of lipooligosaccharide, including possible interactions with capsule, affect the ability of meningococcal endotoxin to interact with and activate specific host targets.

Asunto(s)

RESUMEN

Moraxella catarrhalis is a respiratory pathogen responsible for acute bacterial otitis media in children and exacerbation of chronic bronchitis in adults. M. catarrhalis strains are frequently resistant to the bactericidal activity of normal human serum. In order to determine if the lipooligosaccharide (LOS) of M. catarrhalis has a role in serum resistance, the UDP-glucose-4-epimerase (galE) gene was identified, cloned, and sequenced and a deletion/insertion mutation was introduced into M. catarrhalis strain 2951. GalE enzymatic activity, measured in whole-cell lysates, was ablated in M. catarrhalis 2951 galE. Mass spectrometric analysis of LOS isolated with hot phenol-water confirmed that strain 2951 produced a type A LOS. These studies showed that the LOS from 2951 galE had lost two hexose residues due to the galE mutation and that the resultant LOS structure lacked the (Galalpha1-4Galbeta1-4Glc) P(k) epitope found on M. catarrhalis 2951. Wild-type M. catarrhalis 2951 is resistant to complement-mediated serum bactericidal activity. In contrast, a greater than 2-log(10)-unit reduction in CFU occurred after incubation of 2951 galE in either 50 or 25% pooled human serum (PNHS), and CFU in 10% PNHS decreased by about 1 log(10) unit. These studies suggest that the P(k) epitope of the LOS may be an important factor in the resistance of M. catarrhalis to the complement-mediated bactericidal effect of normal human serum.

Asunto(s)

RESUMEN

Sodium dodecyl sulfate-polyacrylamide gel analysis of lipooligosaccharide (LOS) from Neisseria meningitidis has demonstrated considerable microheterogeneity in the variable region of LOS due to the presence of novel glycoforms. As a step toward understanding the basis for the expression of these novel glycoforms, we have examined the LOS structures and UDP-glucose 4-epimerase (epimerase) activity levels in two strains (NMB and MA-1) and their respective galE mutants. Strain NMB was found to have low epimerase activity and to contain multiple glycoforms, some of which appear to contain only glucose sugars. The galE mutant had only the oligoglucose glycoforms. Strain MA-1 had higher epimerase activity at both log and stationary phases (2- and 12.5-fold, respectively) and one glycoform with a putative lactosyl structure. Strain MA-1 galE had two glycoforms that contained one or two glucose residues. To understand the molecular basis for the different epimerase activities, we examined the predicted amino acid sequences of the respective galE open reading frames and determined the relative amounts of GalE protein. We found no significant differences between the predicted amino acid sequence of the GalE protein in NMB and that in MA-1. We observed no significant differences in the level of GalE protein between MA-1 and NMB at exponential or stationary phase. We also observed an 8.2-fold drop in epimerase activity in NMB between the log and stationary phases that was not due to the GalE protein level or low glucose levels.

Asunto(s)

RESUMEN

This is the introduction of chronic cystitis in course of urinary bladder calculi, 12 years after vesicoureteral reflux operation. The nucleus of the calculus (3 x 3 x 4 cm) were unresorbed dexon sutures left after the surgical treatment. Inaccuracy of radiological and ultrasonography examination delayed the settlement of the diagnosis about 18 months, and patient was caused of many months antibacterial therapy. After removal the calculus from the urinary bladder, dysuric disorders abated quickly and than after 8 weeks antibacterial therapy pathological changes in urine analysis disappeared, urine culture was sterile.

Asunto(s)

RESUMEN

Platelet membrane glycoproteins Ib (GPIb) and IIb/IIIa (GPIIb/IIIa) bind soluble von Willebrand factor (vWf) after stimulation with ristocetin (GPIb) or with thrombin or ADP (GPIIb/IIIa). In fluid-phase, vWf does not bind to these platelet receptors without stimulation. In contrast, platelets adhere to solid-phase vWf without stimulation by ristocetin, adenosine diphosphate (ADP), or thrombin, and adhesion increases after stimulation by these agonists. The effect of monoclonal antibodies specific for GPIb (6D1) and GPIIb/IIIa (10E5 and HP1-1D) on platelet adhesion to solid-phase vWF was studied. Adhesion of radiolabeled, washed platelets (with washed red blood cells) aspirated at a constant wall shear rate of 1000 sec-1 through glass capillary tubes coated with purified human vWf was quantified. Unstimulated platelet adhesion was decreased 80% to 90% by blocking either the GPIb site or the GPIIb/IIIa site with 6D1 or 10E5, respectively, or with 6D1 and 10E5 together. Adhesion was not reduced significantly by HP1-1D (anti-GPIIb/IIIa). After stimulation with ADP or thrombin, the platelet adhesion was reduced by prior incubation with saturating concentrations of either 6D1 (61% reduction) or 10E5 (80% reduction), as well as with both 6D1 and 10E5 (80% reduction). After stimulation with ristocetin, the adhesion was reduced with either 6D1 (90% reduction) or 10E5 (90% reduction) or both 6D1 and 10E5 (90% reduction). Prior incubation with HP1-1D had minimal effect on platelet adhesion to vWF after stimulation with thrombin, ADP, or ristocetin.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

RESUMEN

When platelets are stimulated with adenosine diphosphate (ADP), thrombin, or ristocetin, they bind soluble von Willebrand factor (vWF). In contrast, platelets adhere to solid-phase vWF without apparent stimulus. This work characterizes the adhesion of washed human platelets to highly purified solid-phase human vWF. VWF (iodine 125-labeled vWF) was demonstrated to bind in a quantifiable fashion to the internal surfaces of glass capillary tubes, saturating at a surface density of 3.0 mg/ml. The multimeric structure of bound vWF was the same as that of normal vWF. Platelets were washed, labeled with indium 111, and resuspended with washed red blood cells (RBCs) in balanced salt solution containing Ca++, Mg++, and apyrase. The washed platelet RBC suspension was aspirated through capillary tubes to which vWF was adsorbed. Adhesion of platelets to adsorbed vWF was directly dependent on the surface density of vWF. Increasing wall shear rate (100 to 5000 sec-1) produced increasing platelet adhesion to maximum reached at 2500 sec-1. Platelets bound to the solid-phase vWF in an irreversible fashion, and, as demonstrated with scanning electron microscopy, they spread on the surface. When used to stimulate the platelets, ADP, thrombin, and ristocetin all increased the platelet adhesion to solid-phase vWF. ADP- and thrombin-stimulated reactions were inhibited by prior treatment of the platelets with 5'-p-fluorosulfonylbenzoyl adenosine. This inhibitor of ADP binding had no effect on the baseline platelet adhesion reaction (without ADP or thrombin). Adenosine in concentration up to 1 mmol/L failed to inhibit adhesion. The data demonstrate that washed platelets adhere to solid-phase vWF without added agonists, that the reaction is dependent on surface density vWF and wall shear rate, that they bind irreversibly, and that they demonstrate surface spreading. In addition, these platelets can be stimulated to increase their adherence to vWF by using ADP, thrombin, and ristocetin.

Asunto(s)

RESUMEN

Multiple assays have been described which quantify von Willebrand Factor (vWF) in plasma or other specimens. An assay using a modification of the microtitre method(1) is described. Washed platelets fixed in formalin and frozen in dimethylsulfoxide are substrate for the reaction. Platelets, ristocetin and the specimen to be assayed are combined in the wells of a microtitre plate. The reaction is mixed for defined times and agglutination of platelets read visually. By adjusting the concentration of platelets, concentration of ristocetin and time, the sensitivity can be adjusted over a wide range.

Asunto(s)

RESUMEN

We describe a technique for visualizing the multimeric structure of von Willebrand factor that does not require use of radioisotopes. This procedure uses a previously described discontinuous dodecyl sulfate agarose gel electrophoretic method followed by transfer of the protein to a nitrocellulose filter and detection by a double-antibody technique in which the second antibody is conjugated to horseradish peroxidase. In the presence of H2O2 and 4-chloro-1-napthol, the antigen appears as a series of about 15 colored bands. This method results in resolution of the high molecular weight components, does not require the use of radioisotopes, and can be completed in 2 days. It should be useful for diagnostic and investigational studies in clinical and research laboratories.

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

The present study was undertaken to evaluate the effects of 1 alpha-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 on bone remodeling in dogs with osteomalacia induced by vitamin D depletion. To assess the rates of skeletal remodeling, intravital tetracycline labeling and morphometry of surface pattern were employed. Either vitamin D3 derivative accelerated the appositional growth rate, increased the percentage of osteoid seams labeled, and decreased the number, width, and perimeter of new osteoid seams. But the derivatives differed in bone resorbing activity: 1 alpha-hydroxyvitamin D3 increased the number and perimeter of resorption sites whereas 24R,25-dihydroxyvitamin D3 decreased them. Thus the results show that the former is a better bone remodeler while the latter may be useful in treating osteoporosis.

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)