(E)-2-hexenal-induced DNA damage and formation of cyclic 1,N2-(1,3-propano)-2'-deoxyguanosine adducts in mammalian cells

(E)-2-Hexenal (hexenal), a natural flavor compound, acts as directly genotoxic agent and forms cyclic 1,N2-propano adducts with deoxyguanosine. Formation of this adduct in isolated DNA and in cells was studied with a modified 32P-postlabeling procedure including HPLC separation, nuclease P1 enrichment, two-dimensional TLC of adducted nucleotide bisphosphates on PEI-cellulose, and quantification of adduct spots by liquid scintillation counting. Adduct formation with the more reactive crotonaldehyde was included for comparison. Synthesized adducted dG-3'-phosphates served as external standards for identification and quantification. In calf thymus DNA, hexenal (0.2 mM) shows a time dependent formation of adducts, yielding 1.55 pmol/mumol of DNA at 5 h incubation. With crotonaldehyde (0.2 mM) the adduct rate was about 10-fold higher. Hexenal also generated 1,N2-propano-dG adducts in the human lymphoblastoid Namalva cell line (0.2 mM, 1 h, 86 fmol/mumol of DNA) and in primary rat colon mucosa cells (0.4 mM, 30 min, 50 fmol/mumol of DNA). In primary colon mucosa cells from rats and humans, hexenal and crotonaldehyde (0.4 mM, 30 min) induced DNA damage, detected by single cell microgel electrophoresis (comet assay). In primary rat gastric mucosa cells, hexenal was only weakly active, inducing detectable DNA damage in 20% of cells at 0.8 mM concentration. In contrast, primary mucosa cells from rat esophagus were as sensitive as colon cells. After single oral application of hexenal to rats (up to 320 mg/kg body wt) DNA damage was not detectable in gastrointestinal mucosa. Analysis of hexenal in selected flavored foods revealed concentrations up to 14 ppm (0.14 mM) that are comparable to its natural occurrence in some fruits and vegetables (up to 30 ppm). Thus, the concentration range selected for the toxicological studies described here clearly is relevant: Hexenal, at concentrations found in food, exerts genotoxic effects in cells from rat and human gastrointestinal tract.     

The G3 domain of versican inhibits mesenchymal chondrogenesis via the epidermal growth factor-like motifs

Versican is a highly expressed proteoglycan in zones of developing tissues. To investigate whether versican plays a role in cell differentiation, we studied its role in mesenchymal condensation and chondrogenesis. Here we report that a mini-versican gene product inhibits mesenchymal chondrogenesis but not condensation. The mini-versican-treated mesenchymal cultures form fewer, smaller cartilaginous nodules and produced lower levels of link protein and type II collagen. The versican G3 domain alone, but not G1, was sufficient to inhibit mesenchymal chondrogenesis. Deletion of two epidermal growth factor (EGF)-like motifs in the G3 domain abolished the effect of versican. The G3 domain of aggrecan, which does not contain an EGF-like motif, did not inhibit mesenchymal chondrogenesis. We also generated a chimera construct containing the two EGF-like motifs of versican and the G3 domain of aggrecan, and we observed that this chimera construct inhibited chondrogenesis to a lesser extent than did the full-length versican G3 construct. Direct transfection of mesenchymal cells with different constructs produced similar results. Furthermore, treatment with versican antisense oligonucleotides and transfection with a versican antisense construct promoted chondrogenesis. Taken together, our results strongly suggest that versican inhibits mesenchymal chondrogenesis via its EGF-like motifs.     

Prospective evaluation of criteria for microbiological diagnosis of prosthetic-joint infection at revision arthroplasty. The OSIRIS Collaborative Study Group

A prospective study was performed to establish criteria for the microbiological diagnosis of prosthetic joint infection at elective revision arthroplasty. Patients were treated in a multidisciplinary unit dedicated to the management and study of musculoskeletal infection. Standard multiple samples of periprosthetic tissue were obtained at surgery, Gram stained, and cultured by direct and enrichment methods. With reference to histology as the criterion standard, sensitivities, specificities, and likelihood ratios (LRs) were calculated by using different cutoffs for the diagnosis of infection. We performed revisions on 334 patients over a 17-month period, of whom 297 were evaluable. The remaining 37 were excluded because histology results were unavailable or could not be interpreted due to underlying inflammatory joint disease. There were 41 infections, with only 65% of all samples sent from infected patients being culture positive, suggesting low numbers of bacteria in the samples taken. The isolation of an indistinguishable microorganism from three or more independent specimens was highly predictive of infection (sensitivity, 65%; specificity, 99.6%; LR, 168.6), while Gram staining was less useful (sensitivity, 12%; specificity, 98%; LR, 10). A simple mathematical model was developed to predict the performance of the diagnostic test. We recommend that five or six specimens be sent, that the cutoff for a definite diagnosis of infection be three or more operative specimens that yield an indistinguishable organism, and that because of its low level of sensitivity, Gram staining should be abandoned as a diagnostic tool at elective revision arthroplasty.     

In vivo efficacy of silver-coated (Silzone) infection-resistant polyester fabric against a biofilm-producing bacteria, Staphylococcus epidermidis

OBJECTIVE: To study the characteristics of birthweight and gestational age of third trimester fetal deaths which occurred before the onset of labour. DESIGN: Review of computerised confidential perinatal mortality records. Data originated from the 1992 Trent Region Perinatal Mortality Survey. SAMPLE: One hundred and forty-nine antepartum stillbirths of at least 24 weeks of gestation confirmed by early ultrasound scan. Congenital abnormalities and multiple pregnancies were excluded. MAIN OUTCOME MEASURES: Reported causes of stillbirth; weight-for-gestational age centiles based on a standard derived from normal pregnancies; pregnancy characteristics compared with the local maternity population. RESULTS: Of 149 stillbirths, 83 (56%) were preterm and 66 were at term, and the majority (126; 85%) occurred from 31 weeks. Most of the deaths (97; 65%) were reported as 'unexplained' even though post-mortems had been carried out in 60% of all cases. Using a gestational age-specific fetal weight standard derived from normal, term live births, 41% of all cases of stillborn infants were small-for-gestational age (< 10th centile; OR 6.2; 95% CI 3.3-11.5); 39% of which had been classified as unexplained were small for gestational age (OR 5.6; 2.6-12.0). This excess of small stillbirths was most pronounced between 31 and 33 weeks, where the weights of 63% of all stillbirths and 72% of unexplained fetal deaths were < 10th centile. Overall, a higher proportion of preterm (< 37 weeks) than term stillbirths were small for gestational age: 53% vs 26% (OR 3.3; 1.6-6.5). However, at term there were also more subtle differences in weight deficit, with more fetuses with a weight between the 10th and 50th centiles than between 50th and 90th (36 vs 11; OR 3.3; 1.4-7.8). Mothers of pregnancies ending in stillbirth were similar in age, size, parity and ethnic group to mothers of live born babies, but were more likely to be smokers (37 vs 27%, OR 1.6; 1.2-2.3). CONCLUSIONS: Many stillborn babies are small for gestational age. In the absence of significant differences in physiological pregnancy characteristics, this is unlikely to be a constitutional smallness, but represents a preponderance of intrauterine growth restriction. For a full appreciation of the strength of this association, appropriate weight standards and classifications need to be applied in perinatal mortality surveys. Many antepartum stillbirths which are currently designated as unexplained may be avoidable if slow fetal growth could be recognised as a warning sign.     

MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway

Among the three Saccharomyces cerevisiae DNA repair epistasis groups, the RAD6 group is the most complicated and least characterized, primarily because it consists of two separate repair pathways: an error-free postreplication repair pathway, and a mutagenesis pathway. The rad6 and rad18 mutants are defective in both pathways, and the rev3 mutant affects only the mutagenesis pathway, but a yeast gene that is involved only in error-free postreplication repair has not been reported. We cloned the MMS2 gene from a yeast genomic library by functional complementation of the mms2-1 mutant [Prakash, L. & Prakash, S. (1977) Genetics 86, 33-55]. MMS2 encodes a 137-amino acid, 15.2-kDa protein with significant sequence homology to a conserved family of ubiquitin-conjugating (Ubc) proteins. However, Mms2 does not appear to possess Ubc activity. Genetic analyses indicate that the mms2 mutation is hypostatic to rad6 and rad18 but is synergistic with the rev3 mutation, and the mms2 mutant is proficient in UV-induced mutagenesis. These phenotypes are reminiscent of a pol30-46 mutant known to be impaired in postreplication repair. The mms2 mutant also displayed a REV3-dependent mutator phenotype, strongly suggesting that the MMS2 gene functions in the error-free postreplication repair pathway, parallel to the REV3 mutagenesis pathway. Furthermore, with respect to UV sensitivity, mms2 was found to be hypostatic to the rad6Delta1-9 mutation, which results in the absence of the first nine amino acids of Rad6. On the basis of these collective results, we propose that the mms2 null mutation and two other allele-specific mutations, rad6Delta1-9 and pol30-46, define the error-free mode of DNA postreplication repair, and that these mutations may enhance both spontaneous and DNA damage-induced mutagenesis.     

A severe adverse reaction to mefloquine and chloroquine prophylaxis

A 23 year old man with no history of neurological or psychiatric illness ingested three weekly 228 mg doses of mefloquine base (250 mg salt) as malaria prophylaxis while in India. He experienced an increasingly severe adverse reaction after each dose, including symptoms of paranoia, hallucinations, and suicidal ideation. The man discontinued mefloquine and continued malaria prophylaxis with chloroquine. Shortly after the first 300 mg dose of chloroquine base (500 mg chloroquine phosphate salt), symptoms acutely intensified and became debilitating. Severe symptoms persisted for 12 months following the discontinuation of both antimalarial drugs.     

Induction of tumor necrosis factor-alpha and apoptosis in gastric mucosal injury by indomethacin: effect of omeprazole and ebrotidine

BACKGROUND: Inhalation of swine dust causes airway inflammation with influx of inflammatory cells, predominantly neutrophils, into the lungs. A study was undertaken to determine whether or not exposure to swine dust induces release of interleukin 8 (IL-8) into upper and lower airways and how this possible release is related to cellular influx. A further aim was to study the relationship between the inflammatory response and swine dust exposure. METHODS: Thirty one healthy, non-smoking, previously unexposed subjects were exposed to swine dust during three hours work in a swine house. Bronchoalveolar lavage (BAL) was performed two weeks before and 24 hours after the exposure (n = 16). Nasal lavage and acoustic rhinometry were carried out 1-2 hours before and seven hours after the start of the exposure (n = 31). Exposure measurements were performed with personal sampling equipment. RESULTS: The exposure led to 19-fold and 70-fold increases in the neutrophil concentrations in nasal lavage and BAL fluid, respectively (p < 0.001). In BAL, fluid macrophages, lymphocytes and eosinophils increased significantly. The IL-8 levels in BAL fluid increased from < 31.3 ng/l to 63 (43-109) ng/l (median (25-75th percentile), p < 0.001), and in nasal lavage fluid the concentrations increased from 144 (97-227) ng/l to 1064 (864-1437) ng/l (p < 0.001). IL-8 levels showed a significant correlation with the increase in neutrophils in the nasal lavage fluid but not in the BAL fluid. Acoustic rhinometry demonstrated significant swelling of the nasal mucosa. The air concentration of inhalable dust was 23.3 (20.0-29.3) mg/m3, endotoxin 1.3 (1.1-1.4) micrograms/m3, and muramic acid 0.99 (0.78-2.1) microgram/m3. CONCLUSIONS: The concentration of IL-8 increases in BAL fluid and nasal lavage fluid following exposure to swine dust and may be one of the chemoattractants contributing to the recruitment of neutrophils to the nasal cavity and the alveolar space.     

Use of monoclonal antibodies in diagnosis of paracoccidioidomycosis: new strategies for detection of circulating antigens

The precise diagnosis of paracoccidioidomycosis, in most cases, is established by direct methods and indirect immunological tests. The latter method is reliant on the identification of the host's humoral responses, which are usually impaired or absent in patients with severe juvenile forms of the disease and in immunocompromised patients. Determining disease activity or assessing treatment responses by measuring antibody levels is difficult, since antibody titer may remain elevated or persist at stationary levels, even in the presence of clinical improvement. Consequently, there is a need for alternative tests aimed at the identification of circulating antigens. A modification of the standard hybridoma production method was used to raise a panel of murine monoclonal antibodies (MAbs) against the yeast form of Paracoccidioides brasiliensis. Of these, MAb PIB, directed against an 87-kDa determinant, was used to develop an inhibition ELISA (inh-ELISA) capable of detecting as little as 5.8 ng of circulating antigen per ml of serum. Sera from 46 patients with paracoccidioidomycosis or other mycoses and sera from healthy individuals were evaluated by the inh-ELISA; overall sensitivity was 80.4% (37 of 46 paracoccidioidomycosis patients tested positive), and specificity compared with that of normal controls from areas of endemicity was 81.4%. The inh-ELISA detected circulating antigen in 100% of patients with the acute form of paracoccidioidomycosis and in 83.3 and 60% of patients with the chronic multifocal and unifocal forms of paracoccidioidomycosis according to the patients' clinical presentation. These results indicate that the inh-ELISA with MAb PIB is effective in the detection of circulating antigen and that this test may be useful for monitoring responses to treatment and establishing disease prognoses.     

Mechanisms of electrical coupling between pyramidal cells

Direct electrical coupling between neurons can be the result of both electrotonic current transfer through gap junctions and extracellular fields. Intracellular recordings from CA1 pyramidal neurons of rat hippocampal slices showed two different types of small-amplitude coupling potentials: short-duration (5 ms) biphasic spikelets, which resembled differentiated action potentials and long-duration (>20 ms) monophasic potentials. A three-dimensional morphological model of a pyramidal cell was employed to determine the extracellular field produced by a neuron and its effect on a nearby neuron resulting from both gap junctional and electric field coupling. Computations were performed with a novel formulation of the boundary element method that employs triangular elements to discretize the soma and cylindrical elements to discretize the dendrites. An analytic formula was derived to aid in computations involving cylindrical elements. Simulation results were compared with biological recordings of intracellular potentials and spikelets. Field effects produced waveforms resembling spikelets although of smaller magnitude than those recorded in vitro. Gap junctional electrotonic connections produced waveforms resembling small-amplitude excitatory postsynaptic potentials. Intracellular electrode measurements were found inadequate for ascertaining membrane events because of externally applied electric fields. The transmembrane voltage induced by the electric field was highly spatially dependent in polarity and wave shape, as well as being an order of magnitude larger than activity measured at the electrode. Membrane voltages because of electrotonic current injection across gap junctions were essentially constant over the cell and were accurately depicted by the electrode. The effects of several parameters were investigated: 1) decreasing the ratio of intra to extracellular conductivity reduced the field effects; 2) the tree structure had a major impact on the intracellular potential; 3) placing the gap junction in the dendrites introduced a time delay in the gap junctional mediated electrotonic potential, as well as deceasing the potential recorded by the somatic electrode; and 4) field effects decayed to one-half of their maximum strength at a cell separation of approximately 20 micron. Results indicate that the in vitro measured spikelets are unlikely to be mediated by gap junctions and that a spikelet produced by the electric field of a single source cell has the same waveshape as the measured spikelet but with a much smaller amplitude. It is hypothesized that spikelets are a manifestation of the simultaneous electric field effects from several local cells whose action potential firing is synchronized.