Risk of pneumonia recurrence in patients previously hospitalized for pneumonia--a retrospective study (1998-2000)

Although elderly hospitalized patients, irrespective of the cause of hospitalization, are known to be at a high risk of subsequent development of pneumonia, some studies suggest the risk to be even higher in those hospitalized for pneumonia than in those hospitalized for other diseases. The aim of this retrospective study was to determine the association of hospitalization for pneumonia and some other diseases with subsequent pneumonia morbidity and mortality. The risk of recurrent pneumonia in patients hospitalized for pneumonia was investigated. Rehospitalization of pneumonia patients previously hospitalized for the same disease was followed-up and compared with rehospitalization of patients hospitalized for other diseases during the same study period. The study included patients aged overl8, initially hospitalized in 1998 for pneumonia (J12-J18), or for some particular gastrointestinal (K20-K31) and urogenital diseases (N10-N12, N30-N39). All rehospitalizations for pneumonia in nine Zagreb hospitals were followed-up during a 3-year study period (1998-2000). Out of 975 patients followed-up for rehospitalization, 227 (23.3%) had initially been hospitalized for pneumonia, and 748 (76.7%) for other diagnoses. During the 3-year period, 30 patients were rehospitalized for pneumonia, out of which number 22 had initially been hospitalized for pneumonia, yielding a statistically significant difference between the two study groups (chi2 = 34.780, p < 0.001). The mortality directly caused by pneumonia was also significantly higher in the group of patients with the initial diagnosis of pneumonia than in the group of patients with other diagnoses (chi2 = 15.82, p < 0.001).     

Activation mechanism of pro-astacin: role of the pro-peptide,tryptic and autoproteolytic cleavage and importance of precise amino-terminal processing

Astacin (EC 3.4.24.21) is a prototype for the astacin family and for the metzincin superfamily of zinc peptidases, which comprise membrane-bound and secreted enzymes involved in extracellular proteolysis during tissue development and remodelling. Generally, metzincins are translated as pro-enzymes (zymogens), which are activated by removal of an N-terminal pro-peptide. In astacin, however, the mode of zymogen activation has been obscured, since the pro-form does not accumulate in vivo. Here we report the detection of pro-astacin in midgut glands of brefeldin A-treated crayfish (Astacus astacus) by immunoprecipitation and mass spectrometry. We demonstrate that the pro-peptide is able to shield the active site of mature astacin as a transient inhibitor, which is degraded slowly. In vitro studies with recombinant pro-astacin in the absence of another protease reveal a potential of auto-proteolytic activation. The initial cleavage in this autoactivation appears to be an intramolecular event. This is supported by the fact that the mutant E93A-pro-astacin is incapable of autoactivation, and completely resistant to cleavage by mature astacin. However, this mutant is cleaved by Astacus trypsin within the pro-peptide. This probably reflects the in vivo situation, where Astacus trypsin and astacin work together during pro-astacin activation. In a first step, trypsin produces amino-terminally truncated pro-astacin derivatives. These are trimmed subsequently by each other and by astacin to yield the mature amino terminus, which forms a salt-bridge with Glu103 in the active site. The disruption of this salt-bridge in the mutants E103A and E103Q results in extremely heat labile proteins, whose catalytic activities are not altered drastically, however. This supports a concept according to which the linkage of Glu103 to the precisely trimmed amino terminus is a crucial structural prerequisite throughout the astacin family.     

Determination of polychlorinated biphenyl congeners in human milk by gas chromatography-negative chemical ionization mass spectrometry after sample clean-up by solid-phase extraction

This paper describes a simple and efficient procedure for measuring 25 congeners of polychlorinated biphenyls in human milk. The limit of quantitation was 0.1 ng/ml for five less chlorinated congeners (PCB 70, 74, 87, 99,101), and 0.01 ng/ml for the remaining 20 congeners (PCB 77, 105, 118, 126, 128, 138, 151, 153, 156, 169, 170, 180, 183, 187, 191, 194, 205, 206, 208 and 209). Solid phase extraction technology was applied to extract the analytes from the matrix and to remove lipids. Three columns were used sequentially, and they were a Bond Elut C(18), a Sep-Pak Plus NH2 and a Bond Elut PCB cartridge. The instrumental method was gas chromatography-mass spectrometry with negative chemical ionization, and selected ion monitoring mode was used.     

Beta-galactofuranose-containing O-linked oligosaccharides present in the cell wall peptidogalactomannan of Aspergillus fumigatus contain immunodominant epitopes

O-linked oligosaccharide groups ranging from di- to hexasaccharide were beta-eliminated by mild alkaline treatment under reducting conditions from the peptidogalactomannan of Aspergillus fumigatus mycelial cell wall. The resulting reduced oligosaccharides, which were the minor components of the peptidogalactomannan fraction, were fractionated to homogeneity by successive gel filtration and high-performance liquid chromatography. Their primary structures were determined based on a combination of techniques including gas chromatography, ESI-QTOF-MS, 1H COSY and TOCSY, and 1H-13C HMQC NMR spectroscopy and methylation analysis, to be: alpha-Glcp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 5)-beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol and beta-Galf-(1 --> 5)-[beta-Galf-(1 --> 5]3-beta-Galf-(1 --> 6)-Man-ol. The beta-Galf containing oligosaccharides have not been previously described as fungal O-linked oligosaccharides. The peptidogalactomannan is antigenic and was recognized by human sera of patients with aspergillosis when probed by ELISA, but de-O-glycosylation rendered a 50% decrease in its reactivity. Furthermore, when tested in a hapten inhibition test, the isolated oligosaccharide alditols were able to block, on a dose-response basis, recognition between human sera and the intact peptidogalactomannan. The immunodominant epitopes were present in the tetra- and hexasaccharide, which contain a beta-Galf-(1 --> 5)-beta-Galf terminal group. These results suggest that the O-glycosidically linked oligosaccharide chains, despite being the less abundant carbohydrate component of the A. fumigatus peptidogalactomannan, may account for a significant part of its antigenicity, other than the known activity associated with the galactomannan component.     

Clinical case of acral hemorrhagic Darier's disease is not caused by mutations in exon 15 of the ATP2A2 gene

Darier's disease (Dyskeratosis follicularis, DD) is a genetic disorder characterized by pathogenetic changes of keratinization with variant forms of cutaneous phenotype. Recently, it has been showed that Darier's disease cause mutations in the ATP2A2 gene, at 12q24.1. The gene encodes sarco-endoplasmic reticulum calcium ATPase type 2 (SERCA2). Mutations in exon 15 are reported to be the most consistent mutations associated with the acral hemorrhagic type of Darier's disease. By direct sequencing we investigated exon 15 of the ATP2A2 gene in a Croation family in which one member had a hemorrhagic Darier's disease, but did not record any mutation in the family we investigated. Our results show that mutations in exon 15 of the ATP2A2 gene are not a necessary prerequisite for acral hemorrhagic type of Darier's disease. Our finding support the variability of clinical manifestations of Darier's disease and lack of genotype/phenotype consistency.     

Glycoproteomics of N-glycosylation by in-gel deglycosylation and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry mapping: application to congenital disorders of glycosylation

A general strategy for the structural evaluation of N-glycosylation, a common post-translational protein modification, is presented. The methods for the release of N-linked glycans from the gel-separated proteins, their isolation, purification and matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) analysis of their mixtures were optimised. Since many glycoproteins are available only at low quantities from sodium dodecyl sulphate-polyacrylamide gel electrophoresis or two-dimensional gels, high attention was paid to obtain N-glycan mixtures representing their actual composition in human plasma by in-gel deglycosylation. The relative sensitivity of solid MALDI matrices for MS analysis of acidic N-glycans was compared. The most favourable results for native acidic N-glycans were obtained with 2,4,6-trihydroxyacetophenone monohydrate/diammoniumcitrate as a matrix. This matrix provided good results for both neutral and acidic mixtures as well as for methylated N-glycans. In the second part of this paper the potential of such an optimised MS strategy alone or in combination with high pH anion-exchange chromatography profiling for the clinical diagnosis of congenital disorders of glycosylation is presented.     

Mass spectrometry and database search in the analysis of proteins from the fungus Pleurotus ostreatus

Tandem mass spectrometry is a method of choice for rapid analysis in proteomics. Identification and characterization of proteins from organisms with sequenced genomes is today a routine procedure as will be identification of proteins from organisms with unsequenced genomes with new developing tools. Here, we report the use of isotopic labeling with electrospray ionisation (ESI)-tandem mass spectrometry for de novo sequencing in combination with database search taking advantage of different programs for identification of fungal proteins. Using this approach we could identify the proteins of interest. Nevertheless, the identification of a novel protein responsible for the conversion of testosterone into androstenedione was still a difficult task, mostly due to the low homology of steroid transforming enzymes, especially those from microorganisms. Protein p27 was identified as the vanillate O-demethylase oxidoreductase, p33 and p36 as two isoenzymes of malate dehydrogenase, and p45 as citrate synthase. By rechecking the sequences using additional programs it could be shown that the protein p36 has a higher local homology to the steroid-transforming enzyme than to the malate dehydrogenase. Therefore, we assume that p36 is a pluripotent enzyme most probably responsible for the 17beta-hydroxysteroid dehydrogenase activity.     

Clinical importance of significant asimptomatic bacteriuria in newborns and infants during early postnatal period

The aim of the study was to detect newborns at risk for developing renal impairment, and to point out the importance of significant asimptomatic bacteriuria in perinatal period and early infancy. Severe urinary tract anomalies are very often accompanied only by asimptomatic bacteriuria in perinatal period. Three urinalysis ware done after delivery. 212 newborns with significant asimptomatic bacteriuria underwent ultrasound examination, and were followed up to three months. Those with normal findings and with passing bacteriuria in the first 2 months were excluded. Group of 52 newborns underwent radioisotope examination. Frequency of urinary tract anomalies in newborns was 34.6%. Increased risk for renal impairment had children with urinary tract anomalies in close family, urinary tract infection or bacteriuria, EPH gestosis and prepartal symptoms of febrile infection in mother, children with IUGR, strangulated umbilical cord, prolonged jaundice and attacks of peripheral cyanosis in perinatal period.