Carcinogen substrate specificity of human COX-1 and COX-2

The activation of carcinogenic aromatic and heterocyclic amines and benzo[a]pyrene-7,8-diol to intracellular electrophiles by prostaglandin H synthase (COX) is well documented for ovine sources of this enzyme. Here, the arachidonic acid-dependent activation of substrates by human (h)COX-1 and-2 is examined, utilizing recombinant enzymes. The COX-dependent activation of benzidine (BZ), 4-aminobiphenyl, (+)benzo[a]pyrene-7,8-diol, (+)benzo[a]pyrene-7,8-diol, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo [4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), and 4,4'-methylenebis(2-chloroaniline) (MOCA) is assessed by means of COX-catalyzed, covalent DNA binding. The hCOX isozymes activated all substrates tested, activation varied from barely detectable for IQ (0.76 and 1.52 pmol bound/mg DNA for COX-1 and -2, respectively) to a high of 65 and 117 pmol bound/mg DNA for COX-1 and -2, respectively, for the activation of MOCA. BZ, which is an excellent peroxidase substrate, did not exhibit high DNA binding levels in hCOX assays and this phenomenon was found to be due to high levels of binding to protein, which effectively competed with the DNA for binding in the assay. The demonstrated ability of the COX enzymes to activate a variety of environmental and dietary carcinogens indicates a potential role for COX in the activation pathway of aromatic and heterocyclic amines and polycyclic hydrocarbons at extra-hepatic sites during early or late stages of carcinogenesis.     

A new approach to measure single-event related brain activity using real-time fMRI: feasibility of sensory, motor, and higher cognitive tasks

Real-time fMRI is a rapidly emerging methodology that enables monitoring changes in brain activity during an ongoing experiment. In this article we demonstrate the feasibility of performing single-event sensory, motor, and higher cognitive tasks in real-time on a clinical whole-body scanner. This approach requires sensitivity optimized fMRI methods: Using statistical parametric mapping we quantified the spatial extent of BOLD contrast signal changes as a function of voxel size and demonstrate that sacrificing spatial resolution and readout bandwidth improves the detection of signal changes in real time. Further increases in BOLD contrast sensitivity were obtained by using real-time multi-echo EPI. Real-time image analysis was performed using our previously described Functional Imaging in REal time (FIRE) software package, which features real-time motion compensation, sliding window correlation analysis, and automatic reference vector optimization. This new fMRI methodology was validated using single-block design paradigms of standard visual, motor, and auditory tasks. Further, we demonstrate the sensitivity of this method for online detection of higher cognitive functions during a language task using single-block design paradigms. Finally, we used single-event fMRI to characterize the variability of the hemodynamic impulse response in primary and supplementary motor cortex in consecutive trials using single movements. Real-time fMRI can improve reliability of clinical and research studies and offers new opportunities for studying higher cognitive functions.     

Presence of Legionellaceae in warm water supplies and typing of strains by polymerase chain reaction

Outbreaks of Legionnaire''s disease present a public health challenge especially because fatal outcomes still remain frequent. The aim of this study was to describe the abundance and epidemiology of Legionellaceae in the human-made environment. Water was sampled from hot-water taps in private and public buildings across the area of Göttingen, Germany, including distant suburbs. Following isolation, we used polymerase chain reaction in order to generate strain specific banding profiles of legionella isolates. In total, 70 buildings were examined. Of these 18 (26%) had the bacterium in at least one water sample. Legionella pneumophila serogroups 1, 4, 5 and 6 could be identified in the water samples. Most of the buildings were colonized solely by one distinct strain, as proven by PCR. In three cases equal patterns were found in separate buildings. There were two buildings in this study where isolates with different serogroups were found at the same time.     

Purification of recombinant hydantoinase and L-N-carbamoylase from Arthrobacter aurescens expressed in Escherichia coli: comparison of wild-type and genetically modified proteins

Two enzymes, hydantoinase (HyuH) and L-N-carbamoylase (HyuC), are required for the biocatalytic production of natural and unnatural, optically pure L-amino acids starting from D,L-5-monosubstituted hydantoins using the so called 'hydantoinase-method'. For the preparation of immobilized enzymes, which omit several drawbacks of whole cell biocatalysts, purified or at least enriched HyuH and HyuC have to be provided. In order to simplify existing purification protocols several genetically modified derivatives of HyuH and HyuC from Arthrobacter aurescens DSM 3747 have been cloned and expressed in E. coli. A fusion protein consisting of maltose-binding protein (MalE) and HyuH resulted in an enhanced solubility of the hydantoinase, which easily forms inclusion bodies. On the other hand the fusion protein could easily be purified with high yield (76%) by just one chromatographic step (amylose resin) and the complex purification protocol of the wild-type enzyme could therefore be simplified and shortened significantly. Interestingly, the specific activity of the MalE-HyuH fusion protein was as high as the wild-type enzyme despite that the molecular mass was doubled. A second modification of HyuH carrying a histidine-tag was efficiently bound to a metal affinity matrix but inactivated completely during elution from the column at either low pH or in the presence of imidazole. In the case of HyuC, an aspartate-tag has been added to the biocatalyst to allow an integrated purification-immobilization procedure since this enzyme is immobilized efficiently only via its carboxylic groups. The diminished isoelectric point of the Asp-tagged HyuC resulted in a simplified purification procedure. Compared to the wild-type enzyme expressed in E. coli HyuC-Asp6 was shifted off the elution range of the contaminating proteins and higher purification factors were obtained even in the capturing step. In contrast to HyuH, it was possible to purify a L-N-carbamoylase carrying a histidine-tag to apparent homogeneity using immobilized metal affinity chromatography. Therefore, the existing three step purification protocol was reduced to one chromatographic step and the yield of this relatively unstable protein enhanced remarkably.     

Automatic and strategic processing of threat cues in patients with chronic pain: a modified stroop evaluation

OBJECTIVE: The goal of this study was to clarify whether patients with chronic pain selectively attend to syndrome-specific (i.e., pain-related) information and, if so, to determine whether this occurs at the conscious (i.e., strategic) or unconscious (i.e., automatic) level. SETTING: This study was conducted at a tertiary care rehabilitation center. PATIENTS: Thirty-three patients with chronic back and/or neck pain and 33 healthy volunteers matched for age, sex, and education participated in this study. OUTCOME MEASURES: A computerized version of a modified Stroop color-naming task, with unmasked and masked conditions, was used to assess strategic and automatic information processing of words related to sensory pain, affect pain, physical threat, social threat, and neutral themes. RESULTS: A repeated-measures ANOVA indicated that patients with chronic pain but not healthy volunteers had delayed color-naming latencies to both sensory and affect pain words in the unmasked condition. Color-naming latency differences were not evident for other word types in the unmasked condition or for any word types in the masked condition. Correlational and regression analyses indicated that the delayed color-naming latencies to pain words in the unmasked condition observed for the chronic pain patients were, in part, associated with high pain-specific cognitive anxiety and interference and lower levels of anxiety sensitivity. CONCLUSIONS: Individuals with chronic pain selectively process pain-related cues at the strategic level but not at the automatic level. Implications of the findings and future research directions are discussed.     

Interleukin-10 in cutaneous disorders: implications for its pathophysiological importance and therapeutic use

Abstract With its antiinflammatory and immunosuppressive properties interleukin-10 (IL-10) plays a dominant role in several immune reactions including regulatory mechanisms in the skin. The overexpression of this mediator has been reported in some inflammatory dermatoses as well as in various skin tumors. These observations are of importance since they may explain the limitation of hyperinflammatory conditions as in eczemas and erythemas on the one hand and the suppression of an adequate antitumor response and thereby the progression of malignant tumors on the other hand. Moreover, elevated IL-10 expression might contribute to an enhanced risk of development of microbacterial superinfections, a frequent finding in several dermatoses, and might also be involved in the pathogenesis of connective tissue diseases. In contrast, recent studies indicate a relative IL-10 deficiency in psoriasis. Early clinical data from psoriatic patients treated with recombinant human IL-10 suggest the therapeutic potential of this cytokine and underline its impact on the regulation of the skin immune system. Received: 11 October 1999 / Accepted: 19 October 1999     

Evaluation of the Sheffield system for identifying children at risk from unexpected death in infancy. Results from Birmingham and Newcastle upon Tyne

The "at birth" system which is used in Sheffield to identify children likely to die unexpectedly in infancy, was tested retrospectively in Birmingham (83 cases) and in Newcastle upon Tyne (56 cases). The discrimination between cases and age-matched controls was poor in both cities. Analysis of the 8 factors used in the system showed that only 2 maintained significant case/control differences in Birmingham and Newcastle. Further investigation showed that other factors from maternity records showed significant case/control differences in these cities. Although the system used in Sheffield would not be of use in a prospective prevention programme in either Newcastle or Birmingham, the possibility of evolving an "at risk" system which might apply more widely is discussed.     

Head injury in child abuse: evaluation with MR imaging

To evaluate the usefulness of magnetic resonance (MR) imaging in the diagnosis of head injury in child abuse, the authors compared the findings at head MR imaging and computed tomography (CT) in 19 abused children. Subdural hematomas (15 cases), cortical contusions (six cases), and shearing injuries (five cases) were demonstrated to particular advantage with MR imaging. CT remained superior in the detection of subarachnoid hemorrhage. MR imaging appears to be valuable in the assessment of patients with suspected intracranial injury due to child abuse.