Purification, kinetic characterization and involvement of tryptophan residue at the NADPH binding site of xylose reductase from Neurospora crassa

Xylose reductase (XR) from Neurospora crassa was purified to homogeneity and was found to be specific to NADPH (nicotinamide adenine dinucleotide phosphate). The purified enzyme showed M(r) of 60 and 29 kDa by gel filtration and SDS-PAGE indicating the presence of two subunits. The kinetic mechanism of xylose reductase is 'iso-ordered bi bi'. Inactivation of XR by N-bromosuccinimide (NBS) was found to be biphasic with second-order rate constants of 2.5 x 10(2) and 80 M-1S-1 for the fast (kf) and slow phase (ks), respectively. NADPH protected 90% of XR activity against inhibition by NBS. The fluorescence and circular dichroism (CD) studies revealed that inactivation was not due to gross conformational change in the enzyme. Analysis of the modified Stern-Volmer plot indicated that 49% of the tryptophanyl fluorescence was available for quenching which was completely abolished in the presence of NADPH confirming the involvement of tryptophan at the coenzyme binding site. Experimental evidence presented here serves to implicate the involvement of a tryptophan residue at the low-affinity NADPH binding site and the nature of this site has been assessed by using the hydrophobic probe ANS.     

A randomized prospective trial comparing 45 and 90-ml oral sodium phosphate with X-Prep in the preparation of patients for colonoscopy

Incontinentia pigmenti is an uncommon genodermatosis that occurs in female infants. The characteristic dental defects are partial anodontia and the presence of some peg-shaped teeth. This report describes a patient with incontinentia pigmenti who exhibited multiple missing teeth in both arches. The patient had malpositioned teeth and decreased occlusal vertical dimension associated with the missing teeth. Orthodontic treatment was required for prosthetic purposes. The dental anomalies of incontinentia pigmenti and the treatment procedure are presented.     

A novel dipstick developed for rapid Bet v 1-specific IgE detection: recombinant allergen immobilized via a monoclonal antibody to crystalline bacterial cell-surface layers

The incidence of allergy to airborne proteins derived from tree and grass pollen, feces of mites, spores of molds, and pet dander has been increasing over the last decades. Since precise diagnosis is a prerequisite for successful immunotherapy, there is a rising demand for rapid, reliable, and inexpensive screening methods such as dipstick assays. With the purified recombinant major birch-pollen allergen rBet v 1a as model protein, crystalline bacterial cell-surface layers (S-layers) were tested for their applicability as an immobilization matrix for dipstick development. For this purpose, S-layers were deposited on a mechanically stable microporous support, cross-linked with glutaraldehyde, and free carboxylic acid groups of the S-layer protein were activated with carbodiimide. In the present test system, rBet v 1a was immobilized via the monoclonal mouse antibody BIP 1, which, unlike the allergen, is too large to enter the pores of the S-layer lattice, and which therefore formed a closed monolayer on the outermost surface of the crystal lattice. Moreover, BIP 1 is known to modulate IgE binding to the allergen. After incubation of the dipsticks in serum, washing of the reaction zone under tap water, and binding of an anti-IgE alkaline phosphatase conjugate, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium was used as substrate, forming an IgE concentration-dependent colored precipitate on the S-layer surface. The investigation of patient sera previously tested with the CAP system confirmed the specificity of the S-layer-based dipstick assay. Since the dipstick is easy to handle and the whole test procedure takes only 90 min, this test system should be applicable for rapid determination of specific IgE and for first screening in the doctor's practice.     

Minimal within-host dengue models highlight the specific roles of the immune response in primary and secondary dengue infections

In recent years, the within-host viral dynamics of dengue infections have been increasingly characterized, and the relationship between aspects of these dynamics and the manifestation of severe disease has been increasingly probed. Despite this progress, there are few mathematical models of within-host dengue dynamics, and the ones that exist focus primarily on the general role of immune cells in the clearance of infected cells, while neglecting other components of the immune response in limiting viraemia. Here, by considering a suite of mathematical within-host dengue models of increasing complexity, we aim to isolate the critical components of the innate and the adaptive immune response that suffice in the reproduction of several well-characterized features of primary and secondary dengue infections. By building up from a simple target cell limited model, we show that only the innate immune response is needed to recover the characteristic features of a primary symptomatic dengue infection, while a higher rate of viral infectivity (indicative of antibody-dependent enhancement) and infected cell clearance by T cells are further needed to recover the characteristic features of a secondary dengue infection. We show that these minimal models can reproduce the increased risk of disease associated with secondary heterologous infections that arises as a result of a cytokine storm, and, further, that they are consistent with virological indicators that predict the onset of severe disease, such as the magnitude of peak viraemia, time to peak viral load, and viral clearance rate. Finally, we show that the effectiveness of these virological indicators to predict the onset of severe disease depends on the contribution of T cells in fuelling the cytokine storm.     

Detection of antibodies to equine arteritis virus by enzyme linked immunosorbant assays utilizing G(L), M and N proteins expressed from recombinant baculoviruses

On the experience of 73 patients, the authors state their guidelines on the treatment of bone metastases on the cervical spine. Most of the cases on which no vertebral collapse occur neither neurological deficit, radiation therapy and external support are suggested. Surgery is necessary on case of severe bone destruction, collapse with or without subsequent neurological impairment. Anterior excision is considered the best approach, sometimes complemented by posterior stabilisation.     

Hyaluronan in human cerebrospinal fluid

We studied the concentration of hyaluronan in cerebrospinal fluid (CSF) in various diseases and attempted to define its reference interval. A radioassay utilizing cartilage proteins with affinity for hyaluronan was used in determining the concentration of 200 lumbar and 27 ventricular CSF specimens and 11 brain cyst fluids. Molecular weight distributions were determined by gel chromatography and localization in brain tissue by histochemistry. The hyaluronan level of lumbar CSF showed an increase with age; comparatively healthy children had (mean +/- SD) 50 +/- 41 micrograms/L (n = 40) and adults 166 +/- 77 micrograms/L (n = 9); i.e. significantly different values. The highest level was recorded in a patient with meningitis (> 8000 micrograms/L). More than 4000 micrograms/ L was noted in a patient with tumour metastasis in the cerebellum. Significantly elevated levels were especially found with spinal stenosis, head injury and cerebral infarction, but also in inflammatory medical disorders, hydrocephalus and encephalitis. We found no significant increase in multiple sclerosis and some other neurological diseases. Ventricular CSF of adults contained significantly less hyaluronan (53 +/- 73 micrograms/L; n = 16) than lumbar CSF. Hyaluronan in cyst fluids varied from 31 to 25,000 micrograms/L. Weight average molecular weight of hyaluronan in CSF was 2.9-3.0 x 10(5) and in brain tumour cyst fluid 2.4 x 10(6). In search for the origin of hyaluronan in CSF it was found that its concentration in the choroid plexus and leptomeninges was low, but that hyaluronan was accumulated in the superficial layer of the cerebral cortex. Continued screening for hyaluronan in CSF may be valuable in cases of inflammatory diseases, tumours and obstruction to CSF flow.     

Marginal adaptation of resin-bonded light-cured glass ionomers in dentin cavities

PURPOSE: To investigate the marginal adaptation of resin-modified glass ionomer cements in dentin cavities placed with or without additional application of resin bonding systems. MATERIALS AND METHODS: Three resin-modified materials (Fuji II LC, Photac-Fil, Vitremer), one compomer (Dyract) and as reference an adhesively bonded resin composite system (Gluma CPS-Pekafill) were used. Flat peripheral dentin surfaces on human molar teeth were produced by wet grinding on SiC paper. Cylindrical cavities, 3.5 mm wide, were prepared in these dentin areas and restored with the individual materials. Sixty cavities were pretreated and restored as requested by the respective manufacturers. Following water storage of the specimens for 15 minutes or 24 hours, excess was gently removed by wet grinding for microscopic inspection of the marginal area. Additionally, in 30 cavities an experimental one-component adhesive resin system, a proprietary dimethacrylate and HEMA mixture dissolved in acetone, was combined with each of the restoratives for evaluation after 15-minutes water storage. Finally, in six cavities each, Dyract was combined with Prime and Bond 2.0, and Vitremer with Scotchbond Multi-Purpose Plus for assessment after 15 minutes. Maximum marginal gap widths (MGW) were measured. One-way ANOVA by ranks (Kruskal-Wallis-Test) followed by Wilcoxon's Two-Sample test were used to study the statistical difference of MGW among the treatment groups at a rejection level P = 0.05. RESULTS: Neither the conventionally placed material systems nor the restorations in combination with adhesives showed consistently gap-free margins after 15-minute water storage. After 24-hour storage with Vitremer 4 of the 6 restorations were gap-free, whereas with all other materials only perfect margins were registered. There was, however, no significant difference between the groups. Application of the experimental and/or the specific resin bonding agents had no effect on early MGW except for the Photac-Fil group, which was significantly reduced.     

Spirochetes from digital dermatitis lesions in cattle are closely related to treponemes associated with human periodontitis

Digital dermatitis (DD), first described in 1974 by Cheli and Mortellaro (R. Cheli and C. Mortellaro, p. 208-213, in Proceedings of the 8th International Conference on Diseases of Cattle, 1974), is a major problem in diary cows and beef cattle causing significant economic losses worldwide. Lesions are typically found at the volar skin proximal to the heel bulbs. Microscopic examination of biopsies or touch preparations of these lesions revealed a variety of different bacterial morphotypes including significant numbers of spirochetes which often represent the predominant morphotype. We used comparative 16S rRNA sequence analysis to determine the diversity and phylogeny of these hitherto unclassified DD spirochetes. Results indicate that those lesions looked at so far contained at least five spirochetal phylotypes, all clustering within the genus Treponema. Phylotype DDKL-4 was nearly identical (99.4% similarity) to that of a nonpathogenic human treponeme, T. phagedenis. Two phylotypes DDKL-3 and DDKL-13 were closely related to those from treponemes commonly found in human periodontitis lesions, i.e., T. denticola and T. vincetii, exhibiting 95 and 98% similarity, respectively. The other two phylotypes, DDKL-12 and DDKL-20, had no close relatives to any cultivable treponemal species but clustered to previously described group IV oral treponemes. Preliminary analysis using in situ hybridization with fluorescently labeled oligonucleotide probes against smears from DD biopsies revealed that from all lesions analyzed so far, T. denticola-like spirochetes were detected in the highest proportion of all spirochetal morphotypes.