Nitroxide-mediated protection against X-ray- and neocarzinostatin-induced DNA damage.

The stable free radical Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxy) has been shown to protect against X-ray-induced cytotoxicity and hydrogen peroxide- or xanthine oxidase-induced cytotoxicity and mutagenicity. The ability of Tempol to protect against X-ray- or neocarzinostatin (NCS)-induced mutagenicity or DNA double-strand breaks (dsb) was studied in Chinese hamster cells. Tempol (50 mM) provided a protection factor of 2.7 against X-ray-induced mutagenicity in Chinese hamster ovary (CHO) AS52 cells, with a protection factor against cytotoxicity of 3.5. Using the field inversion gel electrophoresis technique of measuring DNA dsb, 50 mM Tempol provides a threefold reduction in DNA damage at an X-ray dose of 40 Gy. For NCS-induced damage, Tempol increased survival from 9% to 80% at 60 ng/mL NCS and reduced mutation induction by a factor of approximately 3. DNA dsb were reduced by a factor of approximately 7 at 500 ng/mL NCS. Tempol is representative of a class of stable nitroxide free radical compounds that have superoxide dismutase-mimetic activity, can oxidize metal ions such as ferrous iron that are complexed to DNA, and may also detoxify radiation-induced organoperoxide radicals by competitive scvenging. The NCS chromophore is reduced by sulfhydryls to an active form. Electron spin resonance (ESR) spectroscopy shows that 2-mercaptoethanol-activated NCS reacts with Tempol 3.5 times faster than does unactivated NCS. Thus, Tempol appears to inactivate the NCS chromophore before a substantial amount of DNA damage occurs.     

Inhibition of human immunodeficiency virus type 1 multiplication by antisense and sense RNA expression.

Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4+ lymphocytes and macrophages and causes AIDS in humans. Retroviral vectors allowing neomycin phosphotransferase (npt) gene expression were engineered to express 5' sequences of HIV-1 RNA in the antisense or sense orientation and used to transform the human CD4+ lymphocyte-derived MT4 cell line. Cells expressing antisense or sense RNA to the HIV-1 tat mRNA leader sequence, as part of the 3' untranslated region of the npt mRNA, remained sensitive to HIV-1 infection. In contrast, resistance to HIV-1 infection was observed in cells expressing antisense RNA to the HIV-1 primer-binding site or to the region 5' to the primer-binding site as part of the 3' region of the npt mRNA. Cells expressing the tat mRNA leader sequence in the sense orientation as a precise replacement of the 5' untranslated region of npt mRNA were also resistant to HIV-1. These results indicate that sense and antisense approaches can be used to interfere with HIV-1 multiplication.     

Influence of trace elements on biogas production from mango processing waste in 1.5 m3 KVIC digesters

Summary The influence of trace elements (Co²⁺, Ni²⁺ and Fe³⁺) in varying concentrations and combination, was studied in 1.5 m³ Khadi & Village Industries Commission (KVIC) digesters for biogas generation from mangopeel. Addition of these trace metals enhanced the biogas yield and methane content moderately, the maximum being with the iron fed digester. The digesters were always found to be stable without much variation in total volatile fatty acids (VFA), pH, total alkalinity and other parameters. A methane content of 62% and biogas yield of 0.49 m³/kg VS added was obtained with 4000 mg/L FeCl₃ supplemented mangopeel fed digester as compared to control having biogas yield of 0.22 m³/kg VS added with a methane content of about 48–50%.     

Adenine photodimerization in deoxyadenylate sequences: elucidation of the mechanism through structural studies of a major d(ApA) photoproduct.

The mechanism of the photodimerization of adjacent adenine bases on the same strand of DNA has been elucidated by determining the structure of one of the two major photoproducts that are formed by UV irradiation of the deoxydinucleoside monophosphate d(ApA). The photoproduct, denoted d(ApA)*, corresponds to a species of adenine photodimer first described by P?rschke (P?rschke, D. (1973) J.Am.Chem.Soc. 95, 8440-8446). From a detailed examination of its chemical and spectroscopic properties, including comparisons with the model compound N-cyano-N1-(1-methylimidazol-5-yl)formamidine, it is deduced that d(ApA)* contains a deoxyadenosine unit covalently linked through its C(8) position to C(4) of an imidazole N(1) deoxyribonucleoside moiety bearing an N-cyanoformamidino substituent at C(5). On treatment with acid, d(ApA)* is degraded with high specificity to 8-(5-amino-imidazol-4-yl)adenine whose identity has been confirmed by independent chemical synthesis. It is concluded that the primary event in adenine photodimerization entails photoaddition of the N(7)-C(8) double bond of the 5'-adenine across the C(6) and C(5) positions of the 3'-adenine. The azetidine species thus generated acts as a common precursor to both types of d(ApA) photoproduct which are formed from it by competing modes of azetidine ring fission.     

Distances between functional sites in cardiac sarcoplasmic reticulum (Ca2+ +Mg2+)-ATPase. Inter-lanthanide energy transfer

The high-affinity Ca2+-binding sites of cardiac sarcoplasmic reticulum (Ca2+ +Mg2+)-ATPase have been probed using trivalent lanthanide ions. Non-radiative energy-transfer studies, using luminescent probe Eu3+ as a donor and Nd3+ or Pr3+ as acceptor, were carried out to estimate the distance between two high-affinity Ca2+-binding/transport sites. Eu3+ was excited directly with pulsed laser light and the energy-transfer efficiency to Nd3+ or Pr3+ was measured, under the conditions in which most donor-acceptor pairs occupied the high-affinity Ca2+ sites. The distance between two high-affinity Ca2+ sites is about 0.89 nm. In the presence of ATP the distance between the high-affinity sites is about 0.855 nm, whereas in the presence of adenosine 5'-[beta, gamma-methylene]triphosphate or adenosine 5'-[beta, gamma-imino]triphosphate the distance is about 0.895 nm. To estimate the distance between the high-affinity Ca2+ sites and ATP-binding/hydrolytic site, we have measured the energy-transfer efficiency between Eu3+ and Cr3+-ATP with Eu3+ at the high-affinity Ca2+ sites and Cr3+-ATP at the ATP-binding/hydrolytic site. Our results show that ATP-binding/hydrolytic site is separated by about 2.2 nm from each high-affinity Ca2+ site.     

The cytoskeleton of neurites after microtubule depolymerization

We previously reported a positive correlation between the number of cold-stable microtubules (MTs) remaining after cold treatment of cat sympathetic nerve and the extent to which the original uniform polarity orientation of axonal MTs was recapitulated after rewarming (J cell biol 99 (1984) 1289). We interpreted these data to indicate that cold-stable fragments, part of larger, generally labile MTs, could act as seeds to organize MT assembly in axons. We report here a direct investigation of the form of cold-stable MTs in neurites of PC-12 cells using two-dimensional reconstruction of serial thin sections. Our data provides strong support for our previous interpretation. The number of MTs in cold-treated neurites was 2-3 times as great while the total length of polymer was approximately half that in control neurites. The average length of MTs in cold-treated neurites was 7-10 times lower than in control neurites. We observed that treatments that depolymerize axonal microtubules cause a marked increase in the number of membranous elements within the axoplasm. This may, however, be a non-specific result of an insult to the axon, since such changes have also been observed in severed, regenerating nerve fibres. We observed that neuroblastoma neurites respond to MT-depolymerization stimuli by developing lateral filopodia similar to those observed in chick dorsal root ganglion cells. Ultrastructural observation of detergent-lysed, whole mounted neuroblastoma (Neuro 2A) cells indicated that the cytoskeleton remaining after MT depolymerization splayed out perpendicular to the long axis of the neurite. That is, we were able to observe many more cytoskeletal 'ends' after MT depolymerization. The concomitant production of filopodia and the splaying of the cytoskeleton after MT depolymerization supports the hypothesis put forward by Wessels et al. (Exp cell res 117 (1978) 335) that the presence or absence of cytoskeletal ends regulates which region of the cell surface is involved in motile behaviour.     

Formation of superoxide and hydroxyl radicals from 1-methyl-4-phenylpyridinium ion (MPP+): reductive activation by NADPH cytochrome P-450 reductase

Formation of free radical intermediates from 1--methyl-4-phenylpyridinium ion(MPP+) has been studied using spin-trapping techniques. Incubation of MPP+ with purified NADPH cytochrome P-450 reductase and NADPH under anaerobic conditions failed to produce any detectable radical intermediates. However, in the presence of air and a spin-trap, a significant stimulation of superoxide and hydroxyl radicals was detected. Formation of these toxic radicals from MPP+ was inhibited by superoxide dismutase, catalase, and ethanol. Under identical conditions, however, considerably less of these radicals were formed with MPP+ in comparison to paraquat, a lung toxin containing two pyridinium moieties.     

Characterization of a two-signal-dependent, Ia+ mononuclear phagocyte progenitor subpopulation that is sensitive to inhibition by ferritin

Evidence is presented that the ferritin-inhibitable, Ia+ monocyte progenitor in murine marrow requires two signals for stimulation of clonal proliferation. Escherichia coli K235 lipopolysaccharide (LPS) at 0.1 ng/ml enhanced macrophage colony formation by 25 to 70% in murine marrow cultures stimulated with colony-stimulating factor (CSF-1). The progenitors which responded to LPS and CSF-1 represented a distinct subpopulation. Pretreatment of marrow cells with complement plus anti-Ia, anti-H2, anti-asialo GM1, and anti-Mac-1 antibodies specifically depleted the two-signal-requiring progenitors. In addition, the same progenitors were depleted by preincubation with hydroxyurea, indicating that these cells were in cell cycle when removed from the marrow. When compared with the quiescent progenitors, the Ia+, cycling cells were more sensitive to the antiproliferative effects of interferon alpha/beta but were more resistant to inhibition by E prostaglandins. Pretreatment with T cell-specific antibodies and complement specifically enhanced cloning of quiescent progenitors without affecting cloning of the Ia+, cycling subpopulation. Moreover, rat liver ferritin at 10(-8) to 10(-10) M specifically inhibited clonal proliferation of the Ia+ progenitors. Finally, the requirement for LPS as the additional stimulant could be replaced by the addition of haplotype-specific anti-Ia antibody to CSF-stimulated cultures. In contrast to LPS, anti-IA was competitive with inhibitory ferritin in clonal proliferation of the Ia+ progenitors. The significance of these observations in regulation of monocytopoiesis is discussed.     

Adrenal-mediated depression of N-acetyltransferase activity and melatonin levels in the rat pineal gland

N-acetyltransferase (NAT) is believed to be the rate-limiting enzyme in the synthesis of melatonin from serotonin in the pineal gland. Norepinephrine released from sympathetic nerve endings within the pineal gland stimulates NAT activity and, therefore, melatonin synthesis. When an animal is subjected to a stressful stimulus, it would be expected that the increase in plasma stimulus, it would be expected that the increase in plasma catecholamines originating from the adrenal medulla and/or the sympathetic nervous system would result in a stimulation of pineal NAT activity. Adult male rats were given a 1.5cc injection of physiological saline subcutaneously into the back leg. Compared to non-injected controls, animals stressed in this manner were shown to have significantly lower pineal melatonin content 10 min after the saline injection late in the light phase of the light/dark cycle (at 18.30 h-lights on at 07.00 h). To test this more thoroughly, a time course study was conducted during the dark phase (at 02.00 h-5 hours after lights out) when pineal NAT activity and melatonin levels are either increasing or elevated. NAT activity and melatonin levels in the pineal were significantly depressed in stressed animals as compared to controls by 10 min after the saline injection, and remained so until 60 min after injection. By 90 min they had returned to control values. In the next study the nighttime response of the pineal to stress was compared in intact and adrenalectomized rats. Adrenalectomy prevented the changes in NAT activity and melatonin content associated with the saline injection. Some factor, such as a catecholamine or corticosterone from the adrenal, seems to be eliciting the response in the pineal to the saline injection. It is not known if the factor is acting centrally or directly on the pineal gland.