Variability of chromosomally encoded beta-lactamases from Klebsiella oxytoca

The beta-lactamase genes of Klebsiella oxytoca were previously divided into two main groups: bla(OXY-1) and bla(OXY-2). The two beta-lactamase groups were each represented by beta-lactamases with four different pIs. In each group, one form of beta-lactamase is more frequent than the others combined. The beta-lactamase gene of each representative beta-lactamase with a different pI that was not yet sequenced (pIs 5.7, 6.8 [OXY-2], 7.1, 8.2, and 8.8 [OXY-1]) was cloned and sequenced. The susceptibility patterns as well as relative rates and kinetic parameters for beta-lactam hydrolysis revealed that OXY-2 enzymes hydrolyzed several of the beta-lactams that were examined (carbenicillin, cephalothin, cefamandole, ceftriaxone, and aztreonam) at a greater rate than the OXY-1 enzymes did. Comparison of K. oxytoca beta-lactamases with plasmid-mediated extended-spectrum beta-lactamases MEN-1 and TOHO-1 implied that the threonine at position 168 present in OXY-2 beta-lactamase instead of the alanine in OXY-1 could be responsible for its modified substrate hydrolysis. In each group, the beta-lactamase with a variant pI differs from the main form of beta-lactamase by one to five amino acid substitutions. The substrate profile and the 50% inhibitory concentrations revealed that all substitutions differing from the main form of beta-lactamase were neutral except one difference in the OXY-1 group. This substitution of an Ala to a Gly at position 237 increases the hydrolysis of some beta-lactams, particularly aztreonam; decreases the hydrolysis of benzylpenicillin, cephaloridine, and cefamandole, and decreases the susceptibility to clavulanic acid (fivefold increase in the 50% inhibitory concentration).     

Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism

Significant cleavage by hammerhead ribozymes requires activation by divalent metal ions. Several models have been proposed to account for the influence of metal ions on hammerhead activity. A number of recent papers have presented data that have been interpreted as supporting a one-metal-hydroxide-ion mechanism. In addition, a solvent deuterium isotope effect has been taken as evidence against a proton transfer in the rate-limiting step of the cleavage reaction. We propose that these data are more easily explained by a two-metal-ion mechanism that does not involve a metal hydroxide, but does involve a proton transfer in the rate-limiting step.     

Comparison of a radioallergosorbent (RAST) inhibition method and a monoclonal enzyme linked immunosorbent assay (ELISA) for aeroallergen measurement

BACKGROUND: Mouse and rat urinary proteins are potent occupational allergens for exposed personnel. Methods of measuring airborne allergens differ greatly, and reported levels of allergens vary considerably between laboratories. OBJECTIVES: To compare the values obtained using two different methods of allergen detection. METHODS: Air samples were collected in rat rooms in Sweden and the United Kingdom at 2 L/min on to polytetrafluoroethylene (PTFE) filters and extracted in buffer containing 0.5% v/v Tween 20. Airborne rat urinary allergen (RUA) was measured in all samples by both RAST inhibition using a polyclonal human serum pool (UK) and a two monoclonal antibody sandwich ELISA employing antibodies specific for Rat n 1.02 (alpha2u-globulin) (Sweden). RESULTS: The two methods gave values which were correlated (r2 log values = 0.72, P<0.0001), but differed by several orders of magnitude (median [range] ratio of RAST inhibition/ELISA = 316 [7-26(80)]. There was a systematic bias: as the absolute values increased, the difference in the measurements increased. The rat urine standards used were antigenically similar. CONCLUSIONS: A large contrast in RUA values obtained from the two assays was observed in this study. This may be primarily due to methodological differences, but variations in antibody specificities or composition of allergenic epitopes in the air samples may contribute. The results demonstrate that standardization of methods and antibodies is necessary before interlaboratory comparisons can be made.     

Complete spontaneous remission in a patient with metastatic non-small-cell lung cancer. Case report, review of the literature, and discussion of possible biological pathways involved

Spontaneous remission of cancer (SR) is defined as a complete or partial, temporary or permanent disappearance of all or at least some relevant parameters of a soundly diagnosed malignant disease without any medical treatment or with treatment that is considered inadequate to produce the resulting regression. We report the case of a 61-year-old man who presented with extensive metatastic disease five months after pneumonectomy for poorly differentiated large cell and polymorphic lung cancer. A vast metastatic tumour mass of the abdominal wall was confirmed histolologically and there was clinical and radiographic evidence of liver and lung metastases. Eight months later, the patient was operated on for a hernia, which had developed in the inguinal biopsy scar and the surgeon confirmed complete clinical SR of the abdominal wall metastases. Again five months later there was no longer any radiologic evidence of liver and lung metastases. Complete remission has persisted more than five years. Histology of the primary and of the abdominal metastases were reviewed by several independent pathologists. SR is an extremly rare event in lung cancer. This is the first documented case of clinically evident visceral metastases of a bronchiogenic adenocarcinoma developing after complete resection of the primary and then showing complete SR. The epidemiology of SR is reviewed and possible mechanisms involved in SR are discussed.     

Endothelial cell injury caused by Candida albicans is dependent on iron

MRL-lpr/lpr mice spontaneously develop manifestations of autoimmunity including arthritis, vasculitis, and glomerulonephritis. The paramagnetic molecule nitric oxide has been implicated as an effector molecule in initiation and propagation of these inflammatory conditions. In this study, we utilized electron paramagnetic resonance spectroscopy to directly detect nitrosylated protein complexes as products of nitric oxide in whole blood and in kidneys of MRL-lpr/lpr mice. Electron paramagnetic resonance spectra of blood samples from MRL-lpr/lpr mice showed nitrosyl hemoglobin species. Amounts of blood nitrosyl hemoglobin in MRL-lpr/lpr mice were significantly increased as compared to age-matched control mice. Electron paramagnetic resonance spectra of MRL-lpr/lpr kidney tissue exhibited a signal characteristic of a dinitrosyl-iron-dithiolate complex at g approximately 2.04. Formation of nitrosylated nonheme protein in diseased kidneys is associated with development of glomerulonephritis in the autoimmune mice. The presence of nitrosylated nonheme protein indicates the formation of nitric oxide within the kidneys of the diseased mice signifying in situ renal nitric oxide formation.     

Hyperhomocysteinemia induces elastolysis in minipig arteries: structural consequences, arterial site specificity and effect of captopril-hydrochlorothiazide

Hyperhomocysteinemia is a risk factor for arterial diseases, and the deterioration of the arterial elastic structures is one of the possible mechanisms underlying this epidemiological association. The aim of this paper is to quantitatively characterize such structural alterations and to explore their causes in a previous model of dietary induced mild hyperhomocysteinemia in minipigs. After four months, both a morphodensitometrical analysis of the elastic structure and a biochemical analysis of elastin and elastase activities were performed on the infrarenal abdominal aorta (IRAA) and the proximal left interventricular coronary artery (LIVCA) of control (C), hyperhomocysteinemic (H) and captopril-hydrochlorothiazide (Cp-Htz, 25 + 12.5 mg/d)-treated (H+/-Cp) minipigs (n = 8/group). Hyperhomocysteinemia was found to induce an increase in parietal elastolytic metalloproteinase activities. It resulted in opening and enlargement of fenestrae through the medial elastic laminae and in a decrease in medial elastin content (p < 10(-3)), expressed as well as volume density (%) as weight concentration (microg elastin/mg dry tissue). The thickness of the media and its basic lamellar organization was unchanged. The reduction in volume density was more dramatic in LIVCA (H: 4.7 +/- 0.9 vs C: 8.8 +/- 2.4), where it was evenly distributed within the media, than in IRAA (H: 6.7 +/- 1.1 vs C: 9.3 +/- 1.2), where the deep medial layers were less affected. Cp-Htz partly prevented the hyperhomocysteinemia-induced reduction of the medial elastin content in LIVCA (5.7 +/- 1.2) and IRAA (7.9 +/- 1.4). This effect, occurring in the subintimal layers of the media in both arteries but not in the deeper layers, resulted in a less beneficial effect in LIVCA than in IRAA. This result parallels the moderate beneficial therapeutic effect of ACE inhibitors against coronary atherosclerosis in humans. This paper reports for the first time a quantitative analysis of the arterial site-dependent deterioration of the elastic structure caused by mild hyperhomocysteinemia and the involvement of metalloproteinases in this process. These results confirm that the plaque-independent damage to elastic structure previously described in hyperhomocysteinemic-atherosclerotic minipigs was mainly due to homocysteine. This highlights that the metalloproteinase-related elastolysis and the subsequent structural deterioration is one of the major events underlying the epidemiological association between mild hyperhomocysteinemia and arterial diseases.     

Late death of very low birthweight infants

The causes of 59 postneonatal deaths of very low birthweight infants were determined. Bronchopulmonary dysplasia (BPD) was the cause of 19 deaths. It also coexisted in 12/20 deaths from infection and 9/20 deaths from other causes. Improvement will be best achieved by advances in the prevention and treatment of BPD.