Chemical protection and cell-cycle effects on radiation-induced mutagenesis

Chinese hamster ovary cells in the exponential phase of growth were harvested and separated by the method of centrifugal elutriation into subpopulations enriched with up to 95% G1 phase, 70% S phase and 65% G2 + M phase cells. Cell cycle distributions were routinely monitored by flow cytometry. Following elutriation, aliquots of cells from each of the enriched cell fractions were incubated in the presence or absence of 4 mM of 2-[(aminopropyl)amino] ethanethiol (WR-1065) for 30 min at 37 degrees C. The cells were then irradiated with 60Co gamma-rays or fission-spectrum neutrons from the JANUS research reactor. Both cell killing and mutagenesis were determined. Regardless of the radiation quality used, cells enriched in G1 phase were the most sensitive to radiation-induced mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase locus. The relative magnitude of protection exerted by WR-1065 differed for each of the elutriator separated cell populations. The greatest magnitude of protection, however, was observed for G1-enriched populations, regardless of the radiation quality used or the biological end-point tested.     

Expression of tumor-associated surface membrane antigens on marrow CFU-s, CFC-gm, BFU-e, and brain CFU-s

Antiserum raised against a mouse mast cell line (FMP1) reacts with 90% to 100% of spleen colony-forming units (CFU-s), granulocyte/macrophage colony-forming cells (CFC-gm), erythroid burst-forming units (BFU-e), and 15% of nucleated marrow cells, using a complement-dependent cytotoxicity assay. We demonstrated that bone marrow, spleen, or thymus cells are able to absorb this activity from the antiserum. Although mouse brain cells have low reactivity with anti-FMP1 serum, the cytolysis level was reduced to background when antiserum was absorbed with brain cells. In addition, colony formation by marrow CFU-s, CFC-gm, and BFU-e was no longer prevented when the cells were incubated with brain-absorbed anti-FMP1 serum and complement. These findings suggest the presence of brain-associated antigens on CFU-s, CFC-gm, and BFU-e. To test whether a CFU-s accessory cell population in marrow is affected by treatment with anti-FMP1 serum and complement, antibody-treated marrow cells were mixed with large numbers of thymocytes and injected into recipient mice. Colony formation was not altered, indicating that the antiserum reacted directly with antigens on CFU-s and not on CFU-s accessory cells.     

Relation between the radiation sensitivity of the rat thymus and the subpopulation composition of its lymphocytes

The administration to rats of Freund adjuvant and conditions of their keeping in the autumn time reduce the mass and cellularity of the thymus and inhibit cell proliferation therein, the content of lymphocytes with high buoyant density being relatively increased. The indicated changes are accompanied by a two-fold increase in the death rate of thymus cells both after irradiation of rats and following four-hour incubation.     

PAK4 is required for regulation of the cell-cycle regulatory protein p21, and for control of cell-cycle progression

The serine/threonine kinase PAK4 regulates cytoskeletal architecture, and controls cell proliferation and survival. In most adult tissues PAK4 is expressed at low levels, but overexpression of PAK4 is associated with uncontrolled proliferation, inappropriate cell survival, and oncogenic transformation. Here we have studied for the first time, the role for PAK4 in the cell cycle. We found that PAK4 levels peak dramatically but transiently in the early part of G1 phase. Deletion of Pak4 was also associated with an increase in p21 levels, and PAK4 was required for normal p21 degradation. In serum-starved cells, the absence of PAK4 led to a reduction in the amount of cells in G1, and an increase in the amount of cells in G2/M phase. We propose that the transient increase in PAK4 levels at early G1 reduces p21 levels, thereby abrogating the activity of CDK4/CDK6 kinases, and allowing cells to proceed with the cell cycle in a precisely coordinated way.     

A mathematical model of the effects of hypoxia on the cell-cycle of normal and cancer cells

The evolution of the cell-cycle is known to be influenced by environmental conditions, including lack of extracellular oxygen (hypoxia). Notably, hypoxia appears to have different effects on normal and cancer cells. Whereas both experience hypoxia-induced arrest of the G1 phase of the cell-cycle (i.e. delay in the transition through the restriction point), experimental evidence suggests that only cancer cells undergo hypoxia-induced quiescence (i.e. the transition of the cell to a latent state in which most of the cell functions, including proliferation, are suspended). Here, we extend a model for the cell-cycle due to Tyson and Novak (J. Theor. Biol. 210 (2001) 249) to account for the action of the protein p27. This protein, whose expression is upregulated under hypoxia, inhibits the activation of the cyclin dependent kinases (CDKs), thus preventing DNA synthesis and delaying the normal progression through the cell-cycle. We use a combination of numerical and analytic techniques to study our model. We show that it reproduces many features of the response to hypoxia of normal and cancer cells, as well as generating experimentally testable predictions. For example our model predicts that cancer cells can undergo quiescence by increasing their levels of p27, whereas for normal cells p27 expression decreases when the cellular growth rate increases.     

Haematological studies on90Sr-90Y-toxicity: II. Femoral CFU-s kinetics and mitogen response of spleen cells

Summary Ferrokinetic perturbances in peripheral blood of mice treated with⁹⁰Sr-⁹⁰Y were demonstrated previously. This paper deals with the effects of⁹⁰Sr-⁹⁰Y treatment (2.5, 5, and 10 µCi/mouse), on the haemopoietic stem cell compartment and on the immune-status. The frequency and kinetics of haemopoietic stem cells in femoral marrow (determined as colony forming units in spleen, CFU-s) and their haemopoietic efficiency (as gauged by⁵⁹Fe-and¹²⁵IUdR-uptake) were estimated. The responsiveness of splenic lymphocytes of the same animals to mitogens (Con A, PHA, and LPS) were also measured. In all assays striking dose-dependent changes marked either by depressions or overshoots were observed during the first week post-incorporation. These are correlated with the pattern of deposition (primary and secondary) of the radionuclides⁹⁰Sr-⁹⁰Y. Towards the end of the period of observation and presumably thereafter, the dependence on dose disappeared and the values remained subnormal. An exception to this was the response of splenic lymphocytes to mitogens. Much higher reactivity was recorded up to the end. This higher reactivity is attributed to augmented cellular turnover, the newly recruited lymphocytes (in accord with their extreme radiosensitivity), being probably more reactive.     

Scleral anisotropy and its effects on the mechanical response of the optic nerve head

This paper presents a computational modeling study of the effects of the collagen fiber structure on the mechanical response of the sclera and the adjacent optic nerve head (ONH). A specimen-specific inverse finite element method was developed to determine the material properties of two human sclera subjected to full-field inflation experiments. A distributed fiber model was applied to describe the anisotropic elastic behavior of the sclera. The model directly incorporated wide-angle X-ray scattering measurements of the anisotropic collagen structure. The converged solution of the inverse method was used in micromechanical studies of the mechanical anisotropy of the sclera at different scales. The effects of the scleral collagen fiber structure on the ONH deformation were evaluated by progressively filtering out local anisotropic features. It was found that the majority of the midposterior sclera could be described as isotropic without significantly affecting the mechanical response of the tissues of the ONH. In contrast, removing local anisotropic features in the peripapillary sclera produced significant changes in scleral canal expansion and lamina cribrosa deformation. Local variations in the collagen structure of the peripapillary sclera significantly influenced the mechanical response of the ONH.     

Comparison of the cardiac electrophysiological effects between doxazosin and bunazosin

In Langendorff-perfused adult rat heart with constant pressure at 80 mmHg, we found doxazosin, an α₁ adrenoceptor blocker, at 10 μM prolonged PR interval and induced occasional arrhythmia followed by complete inhibition of the sinus rhythm, whereas bunazosin, another α₁-blocker, at same concentration did not. The results of voltage-clamp study showed that, at the concentration of 10 μM, doxazosin inhibited I _Na, I _(Ca,L) , I _to, and Iss without changing I _k1 but bunazosin only inhibited I _(Ca,L) about 30%. Doxazosin also caused markedly negative shift of the I _Na inactivation curve. In current-clamp study, doxazosin prolonged action potential duration in association with the decreased action potential amplitude and upstroke velocity, whereas bunazosin did not. We hypothesize that doxazosin-induced arrhythmia may result from the heterogeneous or different level of I _(Ca,L) blockade of AV nodal tissue. In conclusion, the present study suggests that bunazosin is safer than doxazosin for long-term treatment in view of electrophysiological effect. However, the underlying mechanism of doxazosin induced nodal arrhythmia is still needed to be determined.     

Nonspecific enhancement of mouse antihapten IgE antibody response: Involvement of a T-cell subpopulation and its product for the potentiation

A concomitant administration of Nippostrongylus brasiliensis saline extract (Nb) with dinitrophenylated ovalbumin (DNP-Ov) significantly enhanced the anti-DNP IgE antibody response in mice which had been irradiated and given a combination of spleen and mesenteric lymph node cells from syngeneic, infected donors and cells from DNP-keyhole limpet hemocyanin (DNP-KLH)-primed donors. The treatment of lymphocytes from infected mice with anti-mouse brain-associated θ serum and C abrogated the enhancing activity. The potentiation occurred in mice receiving nylon wool-nonadherent cells but not in mice receiving adherent cells. Challenge with Nb plus DNP-Ov failed to induce potentiation in C3H mice which are known as nonresponders to low doses of Ov, whereas challenge with Nb plus DNP-bovine gamma globulin (BGG) potentiated the response. However, further increase of the enhanced response was not obtained by adding carrier (BGG or Ov)-primed cells to the transferred lymphocyte populations. When a T-independent antigen, DNP-Ficoll, was used for challenge concomitantly with antigen Nb, no potentiation occurred, even though DNP-Ficoll did not give any tolerogenic or suppressive effect on the IgE antibody response to DNP-Nb. An enhancing activity on the IgE class of antibody response but not on the IgG class was observed in supernatants of in vitro culture of lymphocytes from infected mice upon stimulation of the cells with 10 to 50 μg Nb. These results indicate that the potentiation is mediated by Nb specific T cells via a soluble factor(s) that enhances specifically the IgE class of antibody responses but nonspecifically in terms of antigens used for immunization. The results also suggest that the potentiating factor displays its activity in the presence of other T cells reactive to carrier determinants of the challenging antigen but not of cells which already have committed themselves to the carrier and differentiated as helper cells.     

Analysis of the effect of hydroxyurea on stem cell (CFU-s) kinetics

Abstract. Hydroxyurea induces profound changes in the pluripotential haemopoietic stem cell (CFU-s) kinetics. The main feature of these changes is a synchronous entry of resting Go CFU-s into the cell cycle. The analysis of the passage of the CFU-s cohort through the cell cycle has been largely based on the examination of the fraction of CFU-s which synthesize DNA in the S phase of the cell cycle. This analysis has, however, been hampered by the fact that both the sensitivity of the S phase CFU-s to hydroxyurea and their sensitivity in the [³H] thymidine suicide technique vary as the cells pass through the S phase. Methods which overcome these difficulties have been used in the experiments presented in this paper.
It was demonstrated that hydroxyurea kills only about 80% of the S phase CFU-s. The sensitivity to hydroxyurea gradually decreases as the cells approach the middle part of the S phase and increases again as the cells enter the late portions of the S phase.
The degree of CFU-s synchrony at the point of entry into and exit from, the S phase has been established. Mathematical analysis of the available data suggests that CFU-s pass through the S phase with a mean transit time of 4–79 hr (standard deviation, 1.45 hr).
Hydroxyurea, administered in vivo , blocks CFU-s in the late G1 phase. The duration of this G1-S block, induced by a dose of 1000 mg of hydroxyurea per kg body weight, is approximately 2 hr. The CFU-s in the middle of the S phase, which survive hydroxyurea administration, are also blocked in their passage through the S phase. These cells, however, seem to finish the S phase with a delay of approximately 2 hr.     

植物防御反应与动物免疫应答的比较及其对应性初探

动物经过数亿年的进化直到脊椎动物阶段出现了渐为完整的免疫系统。植物在与各种病原的共进化过程中亦发展了自身的防御系统。随着对植物抗病性的概念及植物防御机制的不断认识,人们发现它与动物的免疫应答有着众多的对应性。这些对应性是否表明植物的防御系统与动物的免疫系统在进化上具有同一性,是否表明它们在防御反应上具有类似的机制值得深思 。     

APOBEC3A can activate the DNA damage response and cause cell-cycle arrest

Human apolipoprotein-B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) proteins constitute a family of cytidine deaminases that mediate restriction of retroviruses, endogenous retro-elements and DNA viruses. It is well established that these enzymes are potent mutators of viral DNA, but it is unclear whether their editing activity is a threat to the integrity of the cellular genome. We show that expression of APOBEC3A can lead to induction of DNA breaks and activation of damage responses in a deaminase-dependent manner. Consistent with these observations, APOBEC3A expression induces cell-cycle arrest. These results indicate that cellular DNA is vulnerable to APOBEC3 activity and deregulated expression of APOBEC3A could threaten genomic integrity.     

Comparison between effects of dantrolene and nifedipine on lipopolysaccharide-induced endotoxemia in the anesthetized rats

Intracellular calcium is an important mediator for regulating the cellular response in endotoxemia. In this study, we investigated the effects of dantrolene and nifedipine, two agents of reducing intracellular calcium levels, on bacterial endotoxin (lipopolysaccharide, LPS; 10 mg/kg i.v.)-induced production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) as well as hemodynamic changes in the anesthetized rat. Injection of LPS (i) induced biphasic changes of blood glucose and rectal temperature: an initial increased phase (<180 min after injection of LPS) followed by a decreased phase (at 240 or 360 min), (ii) caused a significant fall in mean arterial blood pressure from 119+/-3 mmHg (at time 0) to 73+/-67 mmHg (at 360 min) with a concomitant increase of heart rate, (iii) resulted in a substantial hyporeactivity to norepinephrine (NE) (1 microg/kg i.v.), (iv) increased plasma nitrate (an indicator of NO formation) in a time-dependent manner, and (v) induced bell-shape changes in plasma TNF-alpha levels which reached a peak at 60 min. Pretreatment of animals with dantrolene (1 mg/kg i.v. at 20 min prior to LPS) or nifedipine (20 microg/kg i.v. infusion for 20 min at 20 min prior to LPS) not only attenuated the delayed circulatory failure (e.g. delayed hypotension and vascular hyporeactivity to NE), but also prevented the overproduction of NO caused by LPS in the rat. However, the prevention of NO overproduction by dantrolene, but not by nifedipine, was associated with an inhibition of TNF-alpha production elicited by LPS. Thus, both dantrolene and nifedipine have beneficial hemodynamic effects, although through different mechanisms, in animals with endotoxic shock.     

The effects of partial and complete denervation on adult newt forelimb blastema cell-cycle parameters

Abstract. The adult newt blastema cell-cycle time (cct) was measured by the percentage of labeled mitoses (PLM) method at the early-bud and mid-bud stages and was found to be 42.9 and 42.7 h, respectively. At both stages, the DNA synthetic phase (S) occupied the majority (75%) of the cct. However, the blastema labeling index (LI) after a 2-h pulse of ³H-thymidine was less than 30% i.e., considerably less than predicted from the ratio of the duration of S over the cct. Compared to that of controls, the PLM plot for partially denervated blastemas exhibited a coincident and equal-sized first peak of labeled mitoses and a coincident but smaller second peak of labeled mitoses. After 24 h of continuous labeling, the LI of control blastemas reached 53%, whereas the LIs of partially denervated and completely denervated blastemas reached only 33% and 20%, respectively. These results are consistent with the view that many cells of adult newt blastemas are not actively progressing through the cell cycle and that the number of noncycling cells is increased by partial or complete denervation. The noncycling cells are probably in the G₁ phase of the cell cycle.     

病原体调控宿主细胞周期致病机制的研究进展

病原体在长期进化中形成多种策略以利于自身增殖,进而导致多种疾病.这些策略包括干扰宿主免疫反应、调控细胞周期与凋亡等.近年来,人们越来越关注病原体通过干扰宿主细胞周期致病的机制,有助于更好了解病原体与宿主的相互作用.本文综述了病原体引起宿主细胞周期停滞在不同时期的机制,特别是一些细菌分泌毒力因子引起宿主细胞周期紊乱并导致...     

Synthesis and structural and pharmacological properties of cyclopropane-based conformationally restricted analogs of 4-methylhistamine as histamine H3/H4 receptor ligands

On the basis of the previous results on a histamine H₄ receptor agonist 4-methylhistamine and a cyclopropane-based conformationally restricted analog CEIC (3) with potent H₃/H₄ receptor antagonistic effect, 4-methylhistamine analogs 4 and 5 of CEIC were designed and synthesized. Compound 4 showed strong affinity (K_i = 38.7 nM) for the H₃ receptor, which was more potent than a well-known H₃ antagonist thioperamide. Stable tautomer and conformation of 3 and 4, which can affect the pharmacological activity, were analyzed by ab initio calculations.