Chromosome modifications induced in mouse spermatogonia through exposure to high-energy neutrons]

The incidence of reciprocal translocations induced in mouse spermatogonia has been studied in CBA mice given X-ray or neutron exposure. Analysis of dividing spermatocytes at diakinesis-first metaphase stage of meiosis shows that in X-irradiated mice there is a linear dose-response relationship. After exposure to fast neutrons the yield of translocations follows a humped curve with a maximum of chromosome exchanges after exposure to 100 rad.     

pH dependence of proton translocation by Escherichia coli.

Proton translocation in spheroplasts from Escherichia coli has been studied in two mutants, one of which expresses cytochrome o and the other cytochrome d as the terminal oxidase. Using the O2 pulse method, the H+/e- ratio of proton translocation associated with cytochrome o was confirmed to be near 2 at neutral pH, but was found to decrease considerably when the medium pH was raised above 8. At high pH there was an increase in H+/OH- permeability of the cell membrane, but this was not sufficient to explain the decline in proton ejection. The pH effect was confined to cytochrome o-linked activity. It was not present when cytochrome d generated the electrochemical proton gradient. This makes it improbable that the Na+/H+ antiporter is responsible. The most likely explanation for our finding is that there is a "slip" in the proton-pumping mechanism of cytochrome o at high pH.     

Temperature dependence of proton permeation through a voltage-gated proton channel

Voltage-gated proton channels are found in many different types of cells, where they facilitate proton movement through the membrane. The mechanism of proton permeation through the channel is an issue of long-term interest, but it remains an open question. To address this issue, we examined the temperature dependence of proton permeation. Under whole cell recordings, rapid temperature changes within a few milliseconds were imposed. This method allowed for the measurement of current amplitudes immediately before and after a temperature jump, from which the ratios of these currents (I_ratio) were determined. The use of I_ratio for evaluating the temperature dependence minimized the contributions of factors other than permeation. Temperature jumps of various degrees (ΔT, −15 to 15°C) were applied over a wide temperature range (4–49°C), and the Q₁₀s for the proton currents were evaluated from the I_ratios. Q₁₀ exhibited a high temperature dependence, varying from 2.2 at 10°C to 1.3 at 40°C. This implies that processes with different temperature dependencies underlie the observed Q₁₀. A novel resistivity pulse method revealed that the access resistance with its low temperature dependence predominated in high temperature ranges. The measured temperature dependence of Q₁₀ was decomposed into Q₁₀ of the channel and of the access resistances. Finally, the Q₁₀ for proton permeation through the voltage-gated proton channel itself was calculated and found to vary from 2.8 at 5°C to 2.2 at 45°C, as expected for an activation enthalpy of 64 kJ/mol. The thermodynamic features for proton permeation through proton-selective channels were discussed for the underlying mechanism.     

Decreased DNA repair rates and protection from heat induced apoptosis mediated by electromagnetic field exposure

In this study, we demonstrate that electromagnetic field (EMF) exposure results in protection from heat induced apoptosis in human cancer cell lines in a time dependent manner. Apoptosis protection was determined by growing HL-60, HL-60R, and Raji cell lines in a 0.15 mT 60 Hz sinusoidal EMF for time periods between 4 and 24 h. After induction of apoptosis, cells were analyzed by the neutral comet assay to determine the percentage of apoptotic cells. To discover the duration of this protection, cells were grown in the EMF for 24 h and then removed for 24 to 48 h before heat shock and neutral comet assays were performed. Our results demonstrate that EMF exposure offers significant protection from apoptosis (P<.0001 for HL-60 and HL-60R, P<.005 for Raji) after 12 h of exposure and that protection can last up to 48 h after removal from the EMF. In this study we further demonstrate the effect of the EMF on DNA repair rates. DNA repair data were gathered by exposing the same cell lines to the EMF for 24 h before damaging the exposed cells and non-exposed cells with H2O2. Cells were allowed to repair for time periods between 0 and 15 min before analysis using the alkaline comet assay. Results showed that EMF exposure significantly decreased DNA repair rates in HL-60 and HL-60R cell lines (P<.001 and P<.01 respectively), but not in the Raji cell line. Importantly, our apoptosis results show that a minimal time exposure to an EMF is needed before observed effects. This may explain previous studies showing no change in apoptosis susceptibility and repair rates when treatments and EMF exposure were administered concurrently. More research is necessary, however, before data from this in vitro study can be applied to in vivo systems.     

Southern-blot analyses of human T-lymphocyte mutants induced in vitro by gamma-irradiation

G0 phase cultures of human peripheral blood T-lymphocytes from a single individual were exposed to 300 rad of gamma-irradiation from a 137Cs source and cultured in vitro for 8 days to allow phenotypic expression. Thioguanine-resistant (TGr) mutants were isolated by a cell cloning assay in microtiter plates. These mutants were studied by Southern blot analysis to define the gross structural alterations in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene by use of an hprt cDNA probe. A similar analysis of the T-cell receptor (TCR) gene rearrangement patterns was employed to define the independent nature of each mutant colony by use of TCR beta and gamma cDNA probes. 74 mutants were isolated in 5 separate experiments. TCR gene rearrangement analysis showed these to represent 24 independent mutations, of which 18 contained hprt structural alterations. These alterations included simple deletions (10/18) as well as more complex rearrangements resulting in molecular weight changes of restriction fragments representing both the 5' and 3' regions of the hprt gene (4/18 and 4/18, respectively). These results demonstrate that gamma-irradiation primarily induces TGr mutations through gross structural alterations in the hprt gene and that these alterations are randomly distributed across the gene. This approach to mutation analysis will provide information on the types of alterations induced by this irradiation, especially the extent of deletions involving the hprt gene. These results also demonstrate the feasibility of employing in vitro exposure of human T-lymphocytes to a single mutagenic agent as an aid to understanding the mechanisms of mutations occurring in vivo in humans.     

Subchronic microcystin-LR exposure increased hepatic apoptosis and induced compensatory mechanisms in mice

Acute lethal cytotoxicity of microcystin-LR (MC-LR), a toxin produced by fresh-water cyanobacteria, has been attributed to protein phosphatases type 1 and type 2A (PP1/PP2A) inhibition and reactive oxygen species (ROS) generation. However, the effects and molecular mechanisms of prolonged, sublethal MC-LR exposure are less known. We studied mice intraperitonealy injected with saline or 25 μg MC-LR/kg for 28 days (every 2 days). MC-LR induced apoptosis in liver and not in kidneys or heart of treated animals. Liver also showed decreased α-tubulin levels (45.56% ± 7.65% of controls) and activation of p38-MAPK and CaMKII pathways (137.93% ± 11.64% and 419.35% ± 67.83% of the control group, respectively). PP1/PP2A activity decreased from 1.82 ± 0.23 (controls) to 0.91 ± 0.98 mU/mg (MC-LR-treated mice); however, no difference in total Ser/Thr phosphatase activity was found between both the groups. The results demonstrated that apoptosis and cytoskeleton disruption contributed to the hepatic cytotoxic effects of subchronic MC-LR administration. These effects occurred in association with sustained activation of signaling cascades and development of compensatory mechanisms to maintain total Ser/Thr phosphatase activity.     

Nuclear apoptosis induced by isolated mitochondria

We isolated and purified mitochondria from mouse livers and spinach leaves.When added into egg extracts of Xenopus laevis,they caused nuclei of mouse liver to undergo apoptotic changes.Chromatin condensation,margination and DNA ladder were observed.After incubating isolated mitochondria in some hypotonic solutions,and centrifuging these mixtures at mgh speed,we got mitochondrial supernatants.It was found that in the absence of cytosolic factor,the supernatant alone was able to induce apoptotic changes in nuclei.The effective components were partly of protein.DNA fragmentation was partly inhibited by caspase inhibitors AC-DEVD-CHO and AC-YVAD-CHO.Meanwhile,caspase inhibitors fully blocked chromatin condensation.Primary characterization of the nuclear endonuclease(s) induced by mitochondrial supernatants was also conducted.It was found that this endonuclease is different from endonuclease G,cytochrome c-induced nuclease,or Ca^2 -activated endonuclease.     

Cell apoptosis induced by carcinogenic metals

Well-documented evidence suggests that environmental and occupational exposure of toxic metals or metal-containing compounds can cause a number of human diseases, including inflammation and cancer, through DNA damage, protein modifications, or lipid peroxidation. This mini-review addresses the mechanisms of cell death induced by some carcinogenic metals, including arsenic (III), chromium (VI) and vanadium (V). A possible contribution of reactive oxygen species to metal-induced cell death is also discussed.     

Synchronized turbo apoptosis induced by cold-shock

In our research on the role of apoptosis in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE), we aim to evaluate the effects of early and late apoptotic cells and blebs on antigen presenting cells. This requires the in vitro generation of sufficiently large and homogeneous populations of early and late apoptotic cells. Here, we present a quick method encountered by serendipity that results in highly reproducible synchronized homogeneous apoptotic cell populations. In brief, granulocytic 32Dcl3 cells are incubated on ice for 2 h and subsequently rewarmed at 37°C. After 30–90 min at 37°C more than 80–90% of the cells become early apoptotic (Annexin V positive/propidium iodide negative). After 24 h of rewarming at 37°C 98% of the cells were late apoptotic (secondary necrotic; Annexin V positive/propidium iodide positive). Cells already formed apoptotic blebs at their cell surface after approximately 20 min at 37°C. Inter-nucleosomal chromatin cleavage and caspase activation were other characteristics of this cold-shock-induced process of apoptosis. Consequently, apoptosis could be inhibited by a caspase inhibitor. Finally, SLE-derived anti-chromatin autoantibodies showed a high affinity for apoptotic blebs generated by cold-shock. Overall, cold-shock induced apoptosis is achieved without the addition of toxic compounds or antibodies, and quickly leads to synchronized homogeneous apoptotic cell populations, which can be applied for various research questions addressing apoptosis.     

Age dependence of bovine oocyte activation

The ability of bovine oocytes to undergo parthenogenetic activation using either a Ca⁺⁺-Mg⁺⁺-H⁺ ionophore (A23187) or electric shock was investigated, as a prelude to understanding activation potential following nuclear transfer into ooplasm. Oocytes were collected from slaughterhouse ovaries by aspiration of 1–5-mm follicles. The time of placement into maturation medium was noted, and maturational age (time in culture) measured from that point. After exposure to activating conditions eggs were cultured for a further 12–16 hours, fixed, and stained with aceto-orcein. Oocytes that progressed to telophase or pronuclear formation were considered activated. Concentrations of A23187 ranging from 100 pM to 100 μM showed that 1–100 μM levels resulted in 94–100% activation at 30 hours maturation. Frequency of activation differed from controls (no ionophore) at 100 nM (49%; P < 0.05). With A23187 maximum response occurred between 26 and 30 hours of maturation (77% and 92%, respectively). A short pulse electric shock, capable of causing oocyte membrane fusion, gave similar results relative to maturational age (82% and 90% activation for 26 and 30 hours, respectively). Therefore, maximum response to the two activating stimuli occurred in oocytes at similar maturational ages. Exposure to activating conditions prior to onset of activating ability (18 hours) followed by another exposure at 26 hours showed that the oocytes were still fully able to activate upon reaching maturational activation competence. Because cytochalasin B is present in the medium used for nuclear transfer, oocytes were incubated with cytochalasin B prior to exposure to an activating stimulus. Frequency of activation was similar to the control treatment (61% and 73%). The effect of mechanical stress of cytoplasm removal and replacement by electrofusion on activation was also not significant. Overall, maturational age of the oocyte was the main determinant of activation ability.     

低氧暴露诱导肌萎缩发生的机制探讨

高海拔地区常伴随着诸多不利环境因素,使初上高原者处于应激状态,对机体代谢系统产生一系列不良影响,其中之一就是诱发骨骼肌萎缩。肌萎缩是一种肌肉功能减退的反应,表现为肌纤维横截面积减小,肌纤维类型转变和肌肉力量、耐力下降。高原环境造成的肌萎缩与低氧环境密切相关。高原训练是竞技体育中一种提高氧运输能力、心脏供血能力及最大摄氧量的有效训练方式,但此过程中造成的骨骼肌质量丢失会影响运动员力量和耐力的发挥,不利于运动表现;同时,随着高原旅游和低氧减肥的兴起,世居平原大众进入高原后发生的肌量丢失和肌力下降也会影响其健康状态。低氧暴露所诱发的肌萎缩程度由低氧浓度和暴露时长所决定,是一个多器官、多组织参与的整体调控骨骼肌蛋白代谢失衡的过程,且在不同类型的肌纤维中表现不同。但目前关于低氧暴露导致肌萎缩的机制还不完全清楚。因此,本文将就该问题相关研究进展进行综述,以期进一步阐明低氧诱导肌萎缩的生物学机制,从而利用低氧刺激更好地提高运动成绩和服务大众健康。     

JNK and NFκB dependence of apoptosis induced by vinblastine in human acute promyelocytic leukaemia cells

The relationship between the mitogen‐activated protein kinase response, nuclear factor‐κB (NFκB) expression and the apoptosis in human acute promyelocytic leukaemia NB4 cells treated with vinblastine was investigated in this work. Cell viability, subdiploid DNA and cell cycle were analysed by propidium iodide permeability and flow cytometry analyses. Apoptosis was determined by annexin V‐Fluorescein isothiocyanate assays. Western‐blot analysis was used for determination of expression levels of apoptotic factors (p53, Bax and Bcl2), intracellular kinases [serine/threonine‐specific protein kinase, extracellular signal‐regulated kinase and c‐Jun N‐terminal kinase (JNK)], NFκB factor and caspases. Electrophoretic mobility shift assay was usefully applied to study DNA‐NFκB interaction. In NB4 cells, vinblastine produces alteration of p53 and DNA fragmentation. Vinblastine treatment had an antiproliferative effect via the induction of apoptosis producing Bax/Bcl‐2 imbalance. Vinblastine treatment suppressed NFκB expression and depressed NFκB‐DNA binding activity while maintaining JNK activation that subsequently resulted in apoptotic response through caspase‐dependent pathway. Our study provides a possible anti‐cancer mechanism of vinblastine action on NB4 cells by deregulation of the intracellular signalling cascade affecting to JNK activation and NFκB expression. Moreover, JNK activation and NFκB depression can be very significant factors in apoptosis induction by vinblastine. Copyright © 2015 John Wiley & Sons, Ltd.     

L-carnitine counteracts prepubertal exposure to cisplatin induced impaired sperm in adult rats by preventing germ cell apoptosis

We investigated the possible protective effects of L-carnitine on cisplatin induced prepubertal gonadotoxicity and on adult sperm. Prepubertal 30-day-old male rats were divided randomly into three groups: control (n = 12), cisplatin exposed (n = 16) and carnitine treated after cisplatin exposure (n = 16). Rats in the experimental groups were injected with a single dose of cisplatin. L-carnitine was injected 1 h before cisplatin administration and for the following 3 days for the cisplatin + carnitine group. The rats were sacrificed at 31 or 90 days old and their testes were harvested for morphometric and histopathological analysis. Testes of 31-day-old prepubertal rats were examined for germ cell apoptosis using the TUNEL method and for proliferation using PCNA immunostaining. The morphology, motility, quantity and vitality of sperm in epididymal fluid samples of adult 90-day-old rats also were evaluated. L-carnitine treatment reduced testicular damage and the number of TUNEL positive cells significantly, while the number of PCNA positive cells in the cisplatin + carnitine group increased compared to the cisplatin group. During the adult period, epididymal sperm count and viability were improved in rats treated with L-carnitine before prepubertal cisplatin injection. L-carnitine may reduce late testicular and spermatic damage caused by cisplatin administration to prepubertal rats by inducing germ cell proliferation and preventing apoptosis.     

Time dependence of chromosomal aberrations induced in human and monkey lymphocytes by acute and fractionated exposure to 60Co

Changes in the numbers of peripheral lymphocytes with chromosome aberrations were observed in cynomolgus monkeys after fractionated or acute 60Co irradiation at the same total doses. Immediately after irradiation, the yield of dicentrics decreased when the dose was fractionated but remained constant for 20 to 80 days, depending on the dose, when irradiation was acute. Lymphopenia was greater than expected when the dose was fractionated. The kinetics of the loss of chromosome aberrations and the changes in peripheral lymphocyte count occurring in monkeys after acute irradiation were compared to those observed in accidentally irradiated men.     

Inhibition of apoptosis by menadione on exposure to UVA

Quinones are widely distributed in the environment, both as natural products and as pollutants. This paper reports that one of the simplest quinones, 2-methyl-1,4-naphthoquinone (menadione), effectively inhibited apoptosis in the presence of UVA. Menadione suppressed the apoptosis induced by serum depletion and cell detachment. This effect was significantly enhanced by UVA irradiation. An antioxidant, N-acetylcysteine, completely inhibited the antiapoptotic effects of both menadione itself and menadione plus UVA, and peroxidation of the cells after treatment was observed using a probe to detect the intracellular production of peroxides. By contrast, 2-hydroxy-1,4-naphtoquinone (lawsone) showed no antiapoptotic effect in the presence or absence of UVA. Lawsone is reported not to undergo the redox process that produces reactive oxygen species. These results indicated that intracellular peroxidation contributed to the antiapoptotic effects of both menadione itself and menadione plus UVA. Dysregulation of the apoptotic process is critical to carcinogenesis. The photosensitization of quinone compounds as it relates to the inhibition of apoptosis should be examined in the future.     

Suppression of T-lymphocyte cytotoxicity following exposure to sinusoidally amplitude-modulated fields

Significant inhibition of allogeneic cytotoxicity of the target cell MPC-11 by the murine cytotoxic T-lymphocyte line CTLL-1 was observed when the 4-h cytotoxicity assay was conducted in the presence of a 450-MHz field sinusoidally amplitude-modulated at 60 Hz. Exposure of the effector cells to the field prior to adding them to the target cells in the cytolytic assay resulted in a similar inhibition, suggesting a direct interaction of the field with the cytolytic T lymphocyte. The inhibition was preferentially expressed during the early allogeneic recognition phase. Field-exposed cytolytic cells recovered their full cytolytic capacity in 12.5 h. A differential susceptibility was observed with modulation frequencies from 0 to 100 Hz. Peak suppression occurred at 60 Hz modulation, with progressively smaller effects at 40, 16, and 3 Hz. The unmodulated carrier wave did not affect the cytotoxicity. Effects with 80- and 100-Hz modulation were smaller than at 60 Hz. These results demonstrate an inhibitory but recoverable effect by certain amplitude modulations of weak nonionizing radiation upon the cell-mediated cytolytic immune response.     

Calcium and proton dependence of sarcoplasmic reticulum ATPase

The influence of Ca2+ and H+ concentrations on the sequential reactions of the ATPase cycle was studied by a series of pre-steady state and steady state experiments with sarcoplasmic reticulum vesicles. It is shown that H+ competition with calcium binding results in a reduced population of activated enzyme, which is manifested by a lower level of phosphorylated enzyme intermediate following addition of ATP. Further effects of Ca2+ and H+ are demonstrated on the progression of the phosphoenzyme through the reaction cycle and on the final hydrolytic cleavage of Pi. The overall dependence of steady state ATP flux on Ca2+ and H+ concentrations in leaky vesicles is expressed by a series of curves showing that as the H+ concentration is raised higher Ca2+ concentrations are required to obtain half-maximal ATP fluxes. At saturating Ca2+, maximal ATP fluxes are observed at an intermediate H+ concentration (pH 7.2), while lower levels are obtained as the H+ concentration is reduced (to pH 8) or increased (to pH 6). A preliminary model is then proposed based on the presence of two interacting domains permitting competitive binding of Ca2+ or H+, per each catalytic site undergoing phosphorylation by ATP. The model considers three main states and thirteen substates (depending on the occupancy of the binding sites in each state by Ca2+, H+, or neither) in the progression of the ATP cycle, coupled to transport of Ca2+ and counter transport of H+ in leaky vesicles. Considering the preliminary nature of the model and the experimental scatter, a rather satisfactory agreement is noted between a family of curves generated by theoretical analysis and the ATP flux curves obtained experimentally.