Fatty acid-induced alteration of the porphyrin macrocycle of cytochrome P450 BM3

Surface-enhanced resonance Raman scattering (SERRS) of substrate-free and substrate-bound forms of the P450 domain of cytochrome P450 BM3 are reported and assigned. Substrate-free P450 yields mixed spin heme species in which the pentacoordinate high-spin arrangement is dominant. The addition of laurate or palmitate leads to an increase in high spin content and to an allosteric activation of heme mode v29, which is sensitive to peripheral heme/protein interactions. Differences between laurate and palmitate binding are observed in the relative intensities of a number of bands and the splitting of the heme vinyl modes. Laurate binding to P450 results in different protein environments being experienced by each vinyl mode, whereas palmitate binding produces a smaller difference. The results demonstrate the ability of SERRS to probe substrate/prosthetic group interactions within an active site, at low protein concentrations.     

Neuroblastoma in Europe: differences in the pattern of disease in the UK. SENSE. Study group for the Evaluation of Neuroblastoma Screening in Europe

BACKGROUND: Neuroblastoma is a major contributor to childhood cancer mortality, but its prognosis varies with age and stage of disease, and some tumours regress spontaneously. Urinary screening programmes or clinical examination may detect the disease before symptoms appear, but the benefit of early diagnosis is uncertain. We examined the incidence, pattern, and presentation of neuroblastoma in four European countries. METHOD: Population-based incidence rates were derived for France, Austria, Germany, and the UK. Age, sex, and stage distribution were analysed by Mantel-Haenszel techniques and Poisson regression. The proportion of incidental diagnoses (cases without symptoms found at routine health checks or during investigation of other disorders) and mortality rates were also compared. FINDINGS: Between 1987 and 1991, 1672 cases of neuroblastoma were diagnosed in children under 15 years old (France, 624; Austria, 69; Germany, 493; UK, 486). Age-standardised annual incidence was significantly lower in the UK (10.1/million) than in France (12.5) and Germany (11.4). In the UK a deficit of low-stage disease in infants was accompanied by an excess of stage IV in older children. The UK had significantly fewer incidental diagnoses (8%) than Austria (27%) and Germany (34%). UK mortality rates were significantly higher than German or French rates. INTERPRETATION: In the UK, neuroblastoma diagnosis is delayed, possibly because of a less rigorous system of health checks for children. Although some overdiagnosis occurs in mainland Europe, our data suggest that in the UK some low-stage cases, undetected in infancy, may later present as advanced disease. This finding has implications for screening programmes and organisation of routine surveillance of infant health in the UK.     

Myelin mosaicism and brain plasticity in heterozygous females of a canine X-linked trait

The shaking (sh) pup, an animal model of Pelizaeus-Merzbacher disease, is characterized by severe central nervous system dysmyelination in affected males, and myelin mosaicism in some female heterozygotes as a result of X-linked inactivation. Heterozygous females develop a tremor of varying severity that usually disappears at 4 to 6 weeks, whereas male hemizygotes have severe, generalized tremor that persists throughout life. We have used these two myelin-deficient models to study the potential for recovery with time as reflected by brainstem auditory evoked responses (BAERs). At set time points, the state of myelination in the trapezoid body was studied microscopically. Sequential BAERs demonstrated consistently prolonged interpeak latencies during the period of gross tremor in heterozygotes, with the trend continuing to a lesser extent after tremor cessation. The random nature of X-linked inactivation resulted in variable myelin mosaicism that was reflected in variations in BAER changes within animals in the same litter. In most heterozygotes, the tremor resolved with time, the BAERs returned to near normal, and myelin mosaicism was lost. In contrast, in the affected males, the severity of tremor and lack of recovery was demonstrated by consistent abnormalities in BAER waves at all times studied, and severe and persistent myelin deficiency in the trapezoid body. These findings show that despite the normal tightly programmed temporal development of myelin in the brain in the heterozygous mosaic state, sufficient plasticity persists during the neonatal period for late-stage myelination to occur.     

Gelatinase A (MMP-2) and cysteine proteinases are essential for the degradation of collagen in soft connective tissue

Epidermal growth factor (EGF) is a potent mitogen for normal mouse mammary epithelial cells grown in primary culture. EGF activation of the EGF-receptor (EGF-R) induces intrinsic tyrosine kinase activity which results in EGF-R autophosphorylation and tyrosine phosphorylation of other intracellular substrates involved in EGF-R signal transduction. Genistein and erbstatin are anticancer agents which have been shown to be potent tyrosine kinase inhibitors. However, the effects of these compounds in modulating EGF-dependent normal mammary epithelial cell proliferation is presently unknown. Therefore, studies were conducted to determine the effects of genistein and erbstatin on EGF-dependent proliferation, and EGF-R levels and autophosphorylation in normal mouse mammary epithelial cells grown in primary culture and maintained in serum-free media. Chronic treatment with 6.25-100 microM genistein or 1-16 microM erbstatin significantly decreased EGF-dependent mammary epithelial cell proliferation in a dose-responsive manner. However, the highest doses of genistein (100 microM) and erbstatin (16 microM) were found to be cytotoxic. Additional studies showed that acute treatment with 6.25-400 microM genistein did not affect EGF-R levels or EGF-induced EGF-R autophosphorylation, while acute treatment with 1-64 microM erbstatin caused a slight reduction in EGF-R levels, but had no effect on EGF-dependent EGF-R autophosphorylation in these cells. In contrast, chronic treatment with similar doses of genistein or erbstatin resulted in a large dose-responsive decrease in EGF-R levels, and a corresponding decrease in total cellular EGF-R autophosphorylation intensity. These results demonstrate that the inhibitory effects of chronic genistein and erbstatin treatment on EGF-dependent mammary epithelial cell proliferation is not due to a direct inhibition of EGF-R tyrosine kinase activity, but results primarily from a down-regulation in EGF-R levels and subsequent decrease in mammary epithelial cell mitogenic-responsiveness to EGF stimulation.     

Genetic effects of 1,3-butadiene and associated risk for heritable damage

A summary of the results of the studies conducted in the EU Project "Multi-endpoint analysis of genetic damage induced by 1,3-butadiene and its major metabolites in somatic and germ cells of mice, rats and man" is presented. Results of the project are summarized on the detection of DNA and hemoglobin adducts, on the cytotoxic and clastogenic effects in somatic and germinal cells of mice and rats, on the induction of somatic mutations at the hprt locus of experimental rodents and occupationally exposed workers, on the induction of dominant lethal mutations in mice and rats, and on heritable translocations induced in mice, after exposure to butadiene (BD) or its major metabolites, butadiene monoepoxide (BMO), diepoxybutane (DEB) and butadiene diolepoxide (BDE). The primary goal of this project was to collect experimental data on the genetic effects of BD in order to estimate the germ cell genetic risk to humans of exposure to BD. To achieve this, the butadiene exposure are based on data for heritable translocations and bone marrow micronuclei induced in mice and chromosome aberrations observed in lymphocytes of exposed workers. A doubling dose for heritable translocations in human germ cells of 4900 ppm/h is estimated, which, assuming cumulative BD exposure over the sensitive period of spermatogenesis, corresponds to 5-6 weeks of continuous exposure at the workplace to 20-25 ppm. Alternatively, the rate of heritable translocation induction per ppm/h of BD exposure is estimated to be approximately 0.8 per million live born, compared to a spontaneous incidence of balanced translocations in humans of approximately 800 per million live born. These estimates have large confidence intervals and are only intended to indicate orders of magnitude of human genetic risk. These risk estimates are based on data from germ cells of BD-exposed male mice. The demonstration that clastogenic damage was induced by DEB in preovulatory oocytes at doses which were not ovotoxic implies that additional studies on the response of mammalian female germ cells to BD and its metabolites are needed. The basic assumption of the above genetic risk estimates is that experimental mouse data obtained after BD exposure can be extrapolated to humans. Several points exist in the present report and in the literature which contradict this assumption: (1) the level of BMO-hemoglobin adducts was significantly elevated in BD-exposed workers; however, it was considerably lower than would have been predicted from comparable rat and mouse exposures; (2) the concentrations of the metabolites DEB and BMO were significantly higher in mouse than in rat blood after BD exposure. Thus, while metabolism of BD is qualitatively similar in the two species, it is quantitatively different; (3) no increase of HPRT mutations was shown in 19 workers exposed on average to 1.8 ppm of BD, while in a different population of workers from a US plant exposed on average to 3.5 ppm of BD, a significant increase of HPRT variants was detected; and (4) data from cancer bioassays and cancer epidemiology suggest that rat is a more appropriate model than mouse for human cancer risk from BD exposure. However, the dominant lethal study in rats gave a negative result. At present, we do not know which BD metabolite(s) may be responsible for the genetic effects even though the bifunctional alkylating agent DEB is the most likely candidate for the induction of clastogenic events. Unfortunately, methods to measure DEB adducts in hemoglobin or DNA are only presently being developed. Despite these several uncertainties the use of the mouse genetic data is regarded as a justifiable and conservative approach to human genetic risk estimation given the considerable heterogeneity observed in the biotransformation of BD in humans.     

Behavioral, physiological, and neuroendocrine stress responses and differential sensitivity to diazepam in two Wistar rat lines selectively bred for high- and low-anxiety-related behavior

Two Wistar rat lines, selectively bred for high-anxiety-related behavior (HAB) and low-anxiety-related behavior (LAB) in the elevated plus-maze test, were tested for the susceptibility of their behavioral characteristics to anxiolytic treatment and for their endocrine and physiological reactivity to different stressors. Injection of 1 mg/kg diazepam failed to affect line differences in coping strategy but resulted in a marked (20-fold) decrease in plus-maze anxiety in HAB rats; whereas, the anxiolytic effect was less pronounced in LAB animals. Biotelemetrical measurements revealed that HAB and LAB rats do not significantly differ in their baseline body temperature, locomotor activity, food and water intake, or in stress-induced alterations of the diurnal rhythms in these parameters. However, line differences were found in acute changes in body temperature and locomotor activity following stress exposure, LAB rats responding with a greater, albeit shorter, increase in body temperature and activity than HAB animals. Basal ACTH and corticosterone plasma levels as well as pituitary reactivity to intravenously administered CRH (40 ng/kg) were similar in both lines, although, especially in response to plus-maze exposure, HAB rats tended toward higher ACTH secretion than LAB rats. These data confirm that animals with high or low basal levels of anxiety may be a promising model for studying the mechanisms of action of anxiolytic substances. Nevertheless, the endocrine findings support the notion that the reactivity of the hypothalamo-pituitary-adrenocortical system and anxiety-related behavior can be regulated independently.     

Assessment of accuracy of the Doppler pressure half-time method in the estimation of the mitral valve area immediately after balloon mitral valvuloplasty

AIM: The reliability of Doppler echocardiography in determining the mitral valve area after balloon mitral valvuloplasty has been questioned, as discrepancies were noted between measurements obtained by the pressure half-time method and those derived haemodynamically, immediately following completion of the procedure. Recent investigations, however, have indicated that these discrepancies may be attributable to the over-estimation of the mitral valve area by haemodynamic measurements, caused by the presence of the iatrogenic atrial septal defect complicating transseptal catheterization. The aim of the present study was to further test this hypothesis. METHODS AND RESULTS: Measurements of the mitral valve area by the Doppler pressure half-time method and the Gorlin formula were obtained and compared in 238 consecutive patients before and immediately after retrograde non-transseptal balloon mitral valvuloplasty, which does not involve puncture and/or dilatation of the inter-atrial septum. No significant difference was found between Doppler- and Gorlin-derived measurements, neither before (1.04 +/- 0.23 vs 1.03 +/- 0.23 cm2, P = ns) nor immediately after (2.14 +/- 0.47 vs 2.12 +/- 0.49 cm2, P = ns) valvuloplasty. Linear regression analysis demonstrated a high degree of correlation between Doppler and Gorlin measurements before (r = 0.778) and after (r = 0.886) the procedure. Good agreement was confirmed by the Bland-Altman method. CONCLUSION: Doppler echocardiography yields accurate measurements of the mitral valve area immediately after retrograde non-transseptal balloon mitral valvuloplasty. This finding supports the hypothesis that the creation of an iatrogenic atrial septal defect during transseptal catheterization may contribute to the poor agreement between Doppler and Gorlin data after balloon mitral valvuloplasty.     

Rational design of novel antimicrobials: blocking purine salvage in a parasitic protozoan

All parasitic protozoa obtain purine nucleotides solely by salvaging purine bases and/or nucleosides from their host. This observation suggests that inhibiting purine salvage may be a good way of killing these organisms. To explore this idea, we attempted to block the purine salvage pathway of the parasitic protozoan Tritrichomonas foetus. T. foetus is a good organism to study because its purine salvage depends primarily on a single enzyme, hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), and could provide a good model for rational drug design through specific enzyme inhibition. Guided by the crystal structure of T. foetus HGXPRTase, we used structure-based drug design to identify several non-purine compounds that inhibited this enzyme without any detectable effect on human HGPRTase. One of these compounds, 4-[N-(3, 4-dichlorophenyl)carbamoyl]phthalic anhydride (referred to as TF1), was selected for further characterization. TF1 was shown to be a competitive inhibitor of T. foetus HGXPRTase with respect to both guanine (in the forward reaction; Ki = 13 microM) and GMP (in the reverse reaction; Ki = 10 microM), but showed no effect on the homologous human enzyme at concentrations of up to 1 mM. TF1 inhibited the in vitro growth of T. foetus with an EC50 of approximately 40 microM. This inhibitory effect was associated with a decrease in the incorporation of exogenous guanine into nucleic acids, and could be reversed by supplementing the growth medium with excess exogenous hypoxanthine or guanine. Thus, rationally targeting an essential enzyme in a parasitic organism has yielded specific enzyme inhibitors capable of suppressing that parasite's growth.     

Acute toxicity of tedisamil, a new potassium channel blocking drug

The acute intravenous toxicity of tedisamil, a new potassium channel blocker, was assessed by infusing it at various rates (1.5 to 10 mg/kg/min.) to ventilated or spontaneously breathing rats subjected to blockade of their peripheral somatic and autonomic nervous systems. The lethal doses for ventilated rats (including pithed animals) were 2-3 times higher than those for spontaneously breathing animals. In spontaneously breathing rats death appeared to be due to respiratory depression which was possibly of central origin since the lethality of tedisamil was not markedly altered by vagotomy, bilateral carotid artery ligation, nor by autonomic nervous system blockade. On the other hand, in all of the artificially ventilated groups, death occurred at higher doses and was related to direct cardiac actions, possibly involving blockade of cardiac potassium and sodium channels. The cardiovascular responses to infusions of tedisamil included tachyarrhythmias, bradycardia, blood pressure changes, increased QRS width and Q-T interval duration. Arrhythmias occurred at sublethal doses and were eliminated by autonomic blockade and pithing. However, the bradycardia, ECG and blood pressure changes induced by tedisamil infusions were unaltered by the various treatments. Thus tedisamil may induce arrhythmias by actions on the autonomic system.