Free radical mechanisms in neocarzinostatin-induced DNA damage

The molecular mechanisms by which the antitumor protein antibiotic, neocarzinostatin, interacts with DNA and causes DNA sugar damage is discussed. Physical binding of the nonprotein chromophore of neocarzinostatin to DNA, involving an intercalative process and dependent on the microheterogeneity of DNA structure, is followed by thiol activation of the drug to a probable radical species. The latter attacks the deoxyribose, especially at thymidylate residues, by abstracting a hydrogen atom from C-5' to generate a carbon-centered radical on the DNA. This nascent form of DNA damage either reacts with dioxygen to form a peroxyl radical derivative, which eventuates in a strand break with a nucleoside 5'-aldehyde at the 5'-end or reacts with the bound drug to form a novel drug-deoxyribose covalent adduct. Nitroaromatic radiation sensitizers can substitute for dioxygen, but the DNA damage products are different. Similarities between the various biological effects of neocarzinostatin and ionizing radiation are reviewed.     

Relation Entre la Teneur en Sucres Réducteurs des Tissus du Tournesol (Helianthus annuus L.) et leur Invasion par Macrophomina phaseolina (Tassi) Goid.

Relation between the amount of reducing sugars in sunflower tissues and their invasion by Macrophomina phaseolina (Tassi) Goid. Reducing sugars (R.S.) were measured with Bernfeld colorimetric method. In the roots, the amount of R.S. increased up to a maximum, then decreased from the beginning of flowering, and remained constant, at a very low level, from the outset of seed maturation. The same evolution was observed, with a delay of one week, in stem bases, but the amounts of R.S. were a little higher. Roots, and thereafter, stems, were invaded by Macrophomina phaseolina only after the minimum level of R.S.was reached. Varietal differences were observed, in a collection of sunflower hybrids, in response to invasion by M. phaseolina. R.S. levels in stem bases were higher in the resistant than in the susceptible hybrids.     

Use of a biotinylated probe and in situ hybridization for light and electron microscopic localization of P 0 mRNA in myelin-forming Schwann cells

Summary A biotinylated P ₀ glycoprotein cDNA was hybridized in situ to aldehyde-fixed vibratome sections and to aldehyde-fixed thin sections of Lowicryl-embedded trigeminal ganglia of 15 day old rats. Alkaline phosphatase and peroxidase detectors were used for light microscopic (LM) studies and peroxidase or colloidal gold were employed for electron microscopic (EM) detection. In both LM and EM sections, probe was found in cytoplasmic areas of myelinforming Schwann cells that were enriched in granular endoplasmic reticulum, demonstrating that these regions contain P ₀ mRNA. Interestingly, P ₀ mRNA tended to cluster in regions close to the developing myelin sheath. Relatively simple methods are here described for EM detection of mRNA with reasonable tissue preservation and high resolution. These methods may be useful for developmental and disease-related studies of specific mRNAs in mammalian tissues.     

Measurement of the transit time for cells through the epidermis and stratum corneum of the mouse and guinea-pig

Abstract A new approach to determine the transit time through the epidermis is presented, involving a gentle washing of the skin surface to collect the loosely attached surface corneocytes. This, it is believed, will be less likely to stimulate the system than tape-stripping or scraping. Radioactively labelled thymidine and iododeoxyuridine have been used to label cells in the basal layer and various labelled amino acids (glycine, cystine and methionine) have been used to label the metabolically viable cell layers (up to and including the granular layer). The resulting changes in surface radioactivity levels have been interpreted to provide a basal to surface transit time of 8–9.5 days for hairless and haired mouse epidermis and about 13.5 days for guinea-pigs. The basal to granular layer transit time, which probably includes some basal layer residence time, is about 4.5 days in the mouse and 8 days in the guinea-pig. The granular to surface time in mice is about 5 days. The results also suggest that when nuclear and cytoplasmic organelles are degraded in the granular layer, material is released that can diffuse rapidly through the stratum corneum to the surface. Some of this can be shown by chromatography to be thymidine. Hence, the stratum corneum is pervious to molecules such as nucleosides. This rapid diffusion outwards through the skin can also be detected shortly after injecting [¹²⁵I]-iododeoxyuridine.     

The effect of irrigation on powdery scab and other tuber diseases of potatoes

In field experiments seed tubers affected with powdery scab cankers were planted and the effect on disease incidence of timing of irrigation and some seed-tuber fungicides was investigated over 3 yr. For 2 yr, irrigation to maintain soil wetter than—20 centibars (—20 kPa) during the first half of the growing season increased disease compared to unirrigated plots. Disease incidence was not affected by irrigation at 2 wk intervals or when applied during the second half of the season. Little disease developed in 1983 even in irrigated plots, probably because of high soil temperatures. None of the fungicides tested gave consistent disease control. Common scab and silver scurf were both decreased by irrigation but in two years, black dot was increased. The relative importance of black dot could increase in irrigated crops where fungicides are used to control silver scurf.     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic prbe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, -lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Stimulation of the hypothalamic-pituitary-adrenal axis and inhibition of growth hormone release via increased central noradrenaline neuronal activity by urethane anaesthesia in the rat: blockade by clonidine

Computerized gas chromatography-mass spectrometry was used to measure precisely the hypothalamic levels of noradrenaline (NA), dopamine and serotonin together with those of their major neuronal metabolites 3,4-dihydroxyphenylethyleneglycol (DHPG), 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in normal male rats 45 min after stimulation of hypothalamic-pituitary-adrenal function by urethane (1.3 g/kg) administration. Urethane treatment resulted in a significant elevation of central noradrenergic neuronal activity (NNA) as assessed from marked rises in hypothalamic DHPG concentrations and the ratio (DHPG/NA). At the same time there was significant stimulation of ACTH and corticosterone release and inhibition of growth hormone release. These hormonal and central effects of urethane (but not anesthesia) were inhibited when the alpha 2-agonist clonidine (150 micrograms/kg) was co-administered. Urethane had no major effect on hypothalamic dopamine or serotonin status. We propose that the release of ACTH and the suppression of growth hormone release following urethane anaesthesia is a result of activation of central NNA and suggest that the hormonal responses are mediated via hypothalamic noradrenergic facilitation of corticotrophin releasing factor and somatostatin release to the anterior pituitary.     

Metabolic activity of bacterial cells enumerated by direct viable count.

The direct viable count (DVC) method was modified by incorporating radiolabeled substrates in microautoradiographic analyses to assess bacterial survival in controlled laboratory microcosms. The DVC method, which permits enumeration of culturable and nonculturable cells, discriminates those cells that are responsive to added nutrients but in which division is inhibited by the addition of nalidixic acid. The resulting elongated cells represent all viable cells; this includes those that are culturable on routine media and those that are not. Escherichia coli and Salmonella enteritidis were employed in the microcosm studies, and radiolabeled substrates included [methyl-3H]thymidine or [U-14C]glutamic acid. Samples taken at selected intervals during the survival experiments were examined by epifluorescence microscopy to enumerate cells by the DVC and acridine orange direct count methods, as well as by culture methods. Good correlation was obtained for cell-associated metabolic activity, measured by microautoradiography and substrate responsiveness (by the DVC method) at various stages of survival. Of the cells responsive to nutrients by the DVC method, ca. 90% were metabolically active by the microautoradiographic method. No significant difference was observed between DVC enumerations with or without added radiolabeled substrate.     

Alternative nonlinear model for estimating second-order rate coefficients for biodegradation.

A modification of the second-order model for biodegradation was derived, applied to an example data set, and shown to be superior for describing the anaerobic biodegradation of p-cresol by an enriched bacterial consortium. The modified model circumvents the no-growth assumption implicit in the use of the second-order rate equation, but still requires the assumption of first-order kinetics over the course of substrate depletion. Violation of the no-growth assumption is particularly important since overestimates of the pseudo-first-order rate coefficient lead to underestimates of the time required for the removal of a xenobiotic chemical from a contaminated environment. Our calculations show that the errors introduced into the pseudo-first-order rate coefficient (and the resulting estimates of the second-order rate coefficient) approach 100% if one doubling occurs in activity over the course of substrate depletion. For an exemplary data set, use of a first-order model resulted in a 100% overestimate of the first-order decay coefficient, which would in turn lead to a corresponding overestimate of the second-order rate coefficient. The modified model we describe is a potential alternative to the pseudo-first-order model for the routine estimation of second-order rate coefficients.