The effect of cystamine on the outcome of dominant lethal mutations and reciprocal translocations in sex cells of mice subjected to gamma-irradiation in different doses]

Protective effect of cystamine (150 mg/kg) against genetic damages induced by gamma-irradiation in germ cells of male mice of CBA strain (at doses of 100, 300, 600 r) was studied. The application of cystamine decreased the frequency of dominant lethal mutations induced by radiation in sperms, spermatids and spermatocytes. The degree of the protective effect of cystamine depended on a radiation dose. The protective effect of cystamine was the highest at the radiation dose of 300 r. It was negligible at the radiation dose of 100 r and was completely absent at the dose of 600 r. Cystamine did not affect the rate of induced reciprocal translocations in the spermatogonia at all the radiation doses used.     

A comparison of chromatin degradation in irradiated normal and tumorous lymphoid cells

Irradiation of mice with doses of 2 and 4 Gy induced extensive chromatin degradation in the thymocytes within 6 hours accompanied by an increase in polydeoxynucleotide (PDN) content (36 and 42 times, respectively). Fifteen hours after irradiation the PDN level was considerably lower, however, still being 4.7 and 14 times the control values after doses of 2 and 4 Gy. The PDN content in control LS/BL lymphosarcoma cells was similar as that in the thymocytes of non-irradiated mice. Unlike in the thymocytes, irradiation of lymphosarcoma cells did induce no statistically significant increase in the PDN level 6 and 15 hours after the irradiation, respectively. It has been reported previously (Matyásová et al. 1973) that chromatin of LS/BL cells degraded similarly as that in the irradiated thymocytes. The results of the present experiments thus provide additional evidence for changes of LS/BL cell properties due to long term cultivation. These cells, however, are still able to react by chromatin fragmentation to nitrogen mustard treatment.     

Suppressing PKC-dependent membrane P2X3 receptor upregulation in dorsal root ganglia mediated electroacupuncture analgesia in rat painful diabetic neuropathy

Painful diabetic neuropathy (PDN) is a common and troublesome diabetes complication. Protein kinase C (PKC)-mediated dorsal root ganglia (DRG) P2X3 receptor upregulation is one important mechanism underlying PDN. Accumulating evidence demonstrated that electroacupuncture (EA) at low frequency could effectively attenuate neuropathic pain. Our previous study showed that 2-Hz EA could relieve pain well in PDN. The study aimed to investigate whether 2-Hz EA relieves pain in PDN through suppressing PKC-mediated DRG P2X3 receptor upregulation. A 7-week feeding of high-fat and high-sugar diet plus a single injection of streptozotocin (STZ) in a dose of 35 mg/kg after a 5-week feeding of the diet successfully induced type 2 PDN in rats as revealed by the elevated body weight, fasting blood glucose, fasting insulin and insulin resistance, and the reduced paw withdrawal threshold (PWT), as well as the destructive ultrastructural change of sciatic nerve. DRG plasma membrane P2X3 receptor level and DRG PKC expression were elevated. Two-hertz EA failed to improve peripheral neuropathy; however, it reduced PWT, DRG plasma membrane P2X3 receptor level, and DRG PKC expression in PDN rats. Intraperitoneal administration of P2X3 receptor agonist αβ-meATP or PKC activator phorbol 12-myristate 13-acetate (PMA) blocked 2-Hz EA analgesia. Furthermore, PMA administration increased DRG plasma membrane P2X3 receptor level in PDN rats subject to 2-Hz EA treatment. These findings together indicated that the analgesic effect of EA in PDN is mediated by suppressing PKC-dependent membrane P2X3 upregulation in DRG. EA at low frequency is a valuable approach for PDN control.     

Utilization of the ferrous sulfate (Fricke) dosimeter for evaluating the radioprotective potential of cystamine: experiment and Monte Carlo simulation

Cystamine, an organic disulfide (RSSR), is among the best of the known radiation-protective compounds and has been used to protect normal tissues in clinical radiation therapy. Recently, it has also proved to be beneficial in the treatment of disorders of the central nervous system in animal models. However, the underlying mechanism of its action at the chemical level is not yet well understood. The present study aims at using the ferrous sulfate (Fricke) dosimeter to quantitatively evaluate, both experimentally and theoretically, the radioprotective potential of this compound. The well-known radiolysis of the Fricke dosimeter by (60)Co γ rays or fast electrons, based on the oxidation of ferrous ions to ferric ions by the oxidizing species (?)OH, HO(2)(?), and H(2)O(2) produced in the radiolytic decomposition of water, forms the basis for our method. The presence of cystamine in Fricke dosimeter solutions during irradiation prevents the radiolytic oxidation of Fe(2+) and leads to decreased ferric yields (or G values). The observed decrease in G(Fe(3+)) increases upon increasing the concentration of the disulfide compound over the range 0-0.1 M under both aerated and deaerated conditions. To help assess the basic radiation-protective mechanism of this compound, a full Monte Carlo computer code is developed to simulate in complete detail the radiation-induced chemistry of the studied Fricke/cystamine solutions. Benefiting from the fact that cystamine is reasonably well characterized in terms of radiation chemistry, this computer model proposes reaction mechanisms and incorporates specific reactions describing the radiolysis of cystamine in aerated and deaerated Fricke solutions that lead to the observable quantitative chemical yields. Results clearly indicate that the protective effect of cystamine originates from its radical-capturing ability, which allows this compound to act by competing with the ferrous ions for the various free radicals--especially (?)OH radicals and H(?) atoms--formed during irradiation of the surrounding water. Most interestingly, our simulation modeling also shows that the predominant pathway in the oxidation of cystamine by (?)OH radicals involves an electron-transfer mechanism, yielding RSSR(?+) and OH(-). A very good agreement is found between calculated G(Fe(3+)) values and experiment. This study concludes that Monte Carlo simulations represent a very efficient method for understanding indirect radiation damage at the molecular level.     

Molecular mechanisms of interphase death of lymphoid cells. Distribution of nuclease attack sites in chromatin and kinetic complexity of DNA in PDN

On the basis of a quantitative correlation between single--and double-helix fragments of different length within PDN, a study was made of the pattern of distribution of the sites, attacked by nuclease, in the chromatin. It was shown that under the effect of ionizing radiation products of enzymic digestion of the chromatin. It was shown that under the effect of ionizing radiation products of enzymic digestion of the chromatin accumulated in thymocytes due to internucleosome degradation were excised from randomly localized genome sites. The analysis of the reassociation curves did not reveal distinctions in the kinetic complexity of the fractions of PDN and total DNA.     

Radioprotective properties of indralin combined with cystamine and mexamine

The study of indralin radioprotective properties at its joint application with cystamine and mexamine was carried out in the experiments on inbred mice and rats. The mice and rats were exposed to whole-body y-irradiation at a dose of 9.0 and 9.5 Gy, correspondingly. A combined parenteral administration ofindralin and cystamine at a dose of 25 mg/kg showed ponentiaton of indralin radioprotective properties up to a level of the ED50 effect versus the absence of or a weak radioprotective effect in the case of their separate application. In the experiments on rats, indralin (50 mg/kg) and mexamine (12 mg/kg) injected intraperitoneally almost completely eliminated the animal mortality from the intestinal syndrome of acute radiation sickness amounting in the control radiation group to 60% on the 7th day after exposure to radiation at a dose of 9.5 Gy. However, at the above conditions, radioprotectors at these doses had a low-level radioprotective action at the onset of the bone marrow syndrome of acute radiation sickness. Combined application of indralin and mexamine at the same doses and at the same conditions led to a radiation protection 50% as high as in the case when radioprotectors were applied separately at a double dose.     

miR-9 Mediates CALHM1-Activated ATP-P2X7R Signal in Painful Diabetic Neuropathy Rats

In this study, we planned to illuminate the mechanisms of the expression and function of CALHM1 in painful diabetic neuropathy (PDN). PDN rat model was constructed. The expression of CALHM1 and miR-9 in rat spinal dorsal horn neurons was detected. The correlation between the level of CALHM1 mRNA and 50 % PWT and the relationship between the expression of CALHM1 and miR-9 in rat spinal dorsal horn neurons were statistically analyzed. The effect of miR-9 and CALHM1 on each other’s expression in PDN rat spinal dorsal horn neurons were tested by qRT-PCR or Western blot. The co-culture system of neurons and glias from PDN rat spinal dorsal horn was constructed. The concentration of calcium and ATP as well as the expression of P2X7 receptor regulated by CALHM1 and miR-9 in PDN rat spinal dorsal horn neurons was measured. The results showed that the expression of CALHM1 was increased in PDN rat compared with controls, while its mRNA level was negatively correlated with 50 % PWT. miR-9, which was also upregulated in the spinal dorsal horn neurons of PDN rats, was positively correlated with the expression of CALHM1. The concentration of calcium and ATP as well as the expression of P2X7 receptor in glias was also increased in PDN rats. These increases could be reverted by inhibiting CALHM1 and/or miR-9. CALHM1 is involved in miR-9-mediated ATP-P2X7 pathway between neurons and glias in PDN rat.     

Characteristics of chromatin breakdown in the rat thymus after exposure to fast neutrons and gamma rays

A comparative study was made of the radiobiological aftereffects of the action of fast neutrons and gamma-rays on lymphoid tissues of rat thymus with a reference to a biochemical criterion of the interphase death of lymphocytes, i.e. the formation of polydeoxynucleotides (PDN). It was shown that the increase in the chromatin degradation was a function of dose of neutron- and gamma-radiation (up to 4 Gy). The dynamics of the PDN formation was similar with both types of radiation, but 4-6 h after neutron irradiation chromatin degradation was higher more pronounced. The RBE of neutrons varied from 3 to 2 with a radiation dose varying from 0.25 to 4 Gy.     

Chemical protection against genetic effect of radiation in male mice

A study was made of the protective effect of some radioprotective agents against dominant lethal mutations (DLM) in postspermatogonial stages and reciprocal translocations (RT) in spermatogonia induced by gamma-radiation. Among the radioprotective agents used, cystaphos, a combination of cystamine and 5-MOT and a mixture of 6 components proved to be most effective against DLM, and cystaphos, gammaphos and cystamine combined with 5-MOT proved effective against RT. The degree of radioprotective efficacy was relatively low. The efficacy of cystamine in protecting against RT was higher with exposure of gonocytes of 18.5-day embryos than spermatogonia of pubertal animals. The degree of the radioprotective effect varied depending on the stage of spermatogenesis, and, in all cases, it was lower than that observed in studies of protection against lethal effects of ionizing radiation.     

Radioprotective properties of indralin combined with cystamine and mexamine

The radioprotective properties of indralin when it is used in combination with cystamine and mexamine are studied in inbred mice and rats. The mice and rats are irradiated with γ rays emitted by ⁶⁰Co at doses of 9.0 and 9.5 Gy, respectively. A combined parenteral administration of indralin and cystamine in mice at doses of 25 mg/kg each is revealed to potentiate the radioprotective properties of indralin up to a level close to the ED₅₀ effect, while the separate application of these drugs in doses of 25 mg/kg each has no or a very weak radioprotective effect. Indralin (50 mg/kg) and mexamine (12 mg/kg) injected intraperitoneally in rats are found to almost completely eliminate the animal mortality caused by gastrointestinal acute radiation syndrome; the mortality in the control radiation group reaches 60% on the seventh day after the animals have been exposed to radiation at a dose of 9.5 Gy. However, if bone-marrow acute radiation syndrome develops under the above condition of super-lethal dose, the radioprotectors have a low radioprotective effect. Under the this condition, the combined application of indralin and mexamine in the same doses has 50% of radioprotective effect reached by applying these radioprotectors separately in double doses.     

Mechanism of thymocyte death in exposure to ultrahigh doses of gamma radiation

A comparative study was made of the morphological and biochemical indices of rat thymus cells after gamma-irradiation with doses of 4-10 Gy (median), 20 Gy (high), and 200-400 Gy (superhigh). It was shown that 4 h after irradiation with superhigh doses the yield of polydeoxynucleotides (PDN) was twice as low as that observed after doses of 4-10 Gy. 24 h after irradiation the amount of the extracted PDN in thymocytes exposed to superhigh doses was markedly larger than that after 4 hours. After all doses applied chromatin degradation occurred at the internucleosome sites in a strict order, the activity of acid and alkaline nucleases being unchanged. A large number of cells have normal nuclear structure 4 h after irradiation (200-400 Gy), as was demonstrated by the electron microscopy data, while in 24 h no intact cells were virtually found in the thymus which correlated with the changes in the PDN yield. The mechanisms of the lymphoid cell death under the effect of different radiation doses are discussed.     

Cystamine attenuates lupus-associated apoptosis of ventricular tissue by suppressing both intrinsic and extrinsic pathways

Cystamine, a disulphide metabolite, has been demonstrated to ameliorate various lupus-associated tissue damages by animal models. However, effects of cystamine on apoptosis of cardiac tissue, a main cardiac damage attributing to lupus, are less obvious. Therefore, we aimed to investigate whether or not cystamine possesses anti-apoptotic effects with emphasis on LV tissue of lupus-prone mice NZB/W-F1. Cystamine treatment was performed by daily intraperitoneal administration. Morphology and apoptotic status of ventricular tissues in the treated mice were assessed by microscopy and TUNEL assay, respectively. Levels of apoptotic biomarkers were determined using immunoblot. Our results revealed that cystamine significantly attenuated the apoptosis of LV tissues in NZB/W-F1 mice, whereas the morphology of the tissues was slightly altered. In addition, cystamine reduced level of Fas and inhibited activation of caspase-8. Cystamine also increased level of Bcl-2 and phosphorylation of Bad, and decreased level of Bad and truncated Bid (tBid). Moreover, level of cytosolic cytochrome c and Apaf-1, and activation of caspase-9 and caspase-3 were suppressed in response to cystamine treatment. In Balb/c mice, as normal control mice, changes in cell morphology and levels of the tested apoptotic components were found insignificant in the LV tissues. These findings indicate that cystamine treatment attenuates apoptosis of LV tissues of NZB/W-F1 mice through suppressing both intrinsic and extrinsic apoptotic pathways. Therefore, cystamine is considered beneficial to alleviating lupus-associated cardiac damages.     

Cystamine inhibits caspase activity. Implications for the treatment of polyglutamine disorders

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an abnormally expended polyglutamine domain. There is no effective treatment for HD; however, inhibition of caspase activity or prevention of mitochondria dysfunction delays disease progression in HD mouse models. Similarly administration of cystamine, which can inhibit transglutaminase, prolonged survival of HD mice, suggesting that inhibition of transglutaminase might provide a new treatment strategy. However, it has been suggested that cystamine may inhibit other thiol-dependent enzymes in addition to transglutaminase. In this study we show that cystamine inhibits recombinant active caspase-3 in a concentration-dependent manner. At low concentrations cystamine is an uncompetitive inhibitor of caspase-3 activity, becoming a non-competitive inhibitor at higher concentrations. The IC(50) for cystamine-mediated inhibition of caspase-3 activity in vitro was 23.6 microm. In situ cystamine inhibited in a concentration-dependent manner the activation of caspase-3 by different pro-apoptotic agents. Additionally, cystamine inhibited caspase-3 activity to the same extent in cell lines stably overexpressing wild type tissue transglutaminase (tTG), a mutant inactive tTG, or an antisense for tTG, demonstrating that cystamine inhibits caspase activity independently of any effects it may have on the transamidating activity of tTG. Finally, treatment with cystamine resulted in a robust increase in the levels of glutathione. These findings demonstrate that cystamine may prolong neuronal survival and delay the onset of HD by inhibiting caspases and increasing the level of antioxidants such as glutathione.     

The effect of cystamine on the survival of mice and endogenous colony-formation in combined radiation-thermal injuries

In experiments on mice it was shown that cystamine exerts its protective action not only against the concurrent effect of ionizing radiation and weak thermal injury but also against the effect of heat (burn) 48 h after irradiation with a midlethal dose (30-day-death decreased from 95 to 20%).     

Chromatin degradation in white blood cells of irradiated rats

A study was made of the dose-time relationship during chromatin degradation in white blood cells of non-irradiated and irradiated rats. There was a linear increase in the release of PDN from leukocytes 1,2 and 3 days after irradiation (1-3 Gy) followed by the deceleration of the chromatin degradation at doses exceeding 3 Gy.     

Cystamine transport in spheroplasts of Saccharomyces cerevisiae

This work is the first demonstration that cystamine is actively accumulated in spheroplasts of Saccharomyces cerevisiae. We have identified and quantitatively determined the transported cystamine in extracts of spheroplasts that have been incubated over different time periods and in the presence of different amounts of cystamine. The method used, already reported in literature for the identification of natural aliphatic polyamines in biological fluids, consists of a derivatization of spheroplast extracts with dabsyl-chloride and subsequent chromatographic analysis in HPLC. Our results show that cystamine accumulation is a function of time, it increases up to 2.5 min then decreases. Transport is inhibited by natural aliphatic polyamines, which, at the same concentration of cystamine (1 mM), cause a decrease in cystamine transport of about 90% for spermidine, 50% for spermine and only 15% for putrescine. Furthermore, transport is energy-dependent as demonstrated by a significant decrease observed in the presence of 2,4-dinitrophenol, ouabain and vanadate. In particular 0.2 mM ouabain causes a decrease of more than 60% in cystamine transport. Our data suggest that cystamine is transported in Saccharomyces cerevisiae spheroplasts via the same polyamine transport system(s) known to be operating in higher eukaryotic cells.     

老年类风湿关节炎患者应用MTX或LEF联合糖皮质激素治疗的效果观察

目的:探讨甲氨蝶呤(MTX)或来氟米特(LEF)联合泼尼松(PDN)对老年类风湿关节炎的疗效。方法:选取112例老年RA患者,按随机数表法将患者分为LEF组(n=28)、MTX组(n=28)、LET+PDN组(n=28)、MTX+PDN组(n=28),治疗3个月后统计四组关节肿胀数、关节压痛数、DAS28评分、VAS评分、RF、晨僵时间,并记录不良反应事件。结果:MTX组和LEF组治疗后各项指标均低于治疗前(P0.05);MTX+PDN组与LEF+PDN组治疗后各项指标均明显低于治疗前(P0.001);MTX+PDN组治疗效果明显优于MTX组(P0.001);LEF+PDN组治疗效果明显优于LEF组(P0.001);MTX+PDN组治疗总有效率为53.57%,高于LEF+PDN组的42.86%,但差异无统计学意义(x~2=2.426,P=0.119)。结论:四组对RA均有治疗效果,联合使用效果更佳,且服药后无严重不良反应,安全性较高。     

Implication of tissue transglutaminase and desmoplakin in cell adhesion mechanism in human epidermis

The distribution patterns of both tissue and keratinocyte transglutaminases (TGase), as well as that of desmoplakin (DP), have been immunohistochemically investigated in human skin cultured in the absence or presence of cystamine and enalapril, two acantholytic agents. In the control samples, tissue TGase is predominantly expressed in lower layers of the epidermis and is located intercellularly. Conversely, in tissues cultured with cystamine or enalapril, a diffuse cytoplasmatic staining was observed. Similarly, DP, detected on the cell membrane in the control, shifts into the cytosol of the keratinocytes following treatment. The distribution pattern of the keratinocyte enzyme in the acantholytic epidermis was identical to that observed in the normal one. Since cystamine and enalapril are TGase inhibitors and DP was shown to act as a TGase substrate in vitro, we suggest that DP and tissue enzyme may participate in cell adhesion at the intraepidermal level.     

Proteomic analysis for the anti‐apoptotic effects of cystamine on apoptosis‐prone macrophage

Increased macrophage vulnerability is associated with progression of systemic lupus erythematosus. Our previous studies have shown that cystamine, an inhibitor of transglutaminase 2 (TG2), alleviated the apoptosis of hepatocyte and brain cell in lupus‐prone mice NZB/W‐F1. In present study, we further investigated the effects of cystamine on apoptosis‐prone macrophages (APMs) in the lupus mice. Using two‐dimensional gel electrophoresis (2‐DE) analysis, we found that cystamine induced a differential protein expression pattern of APM as comparing to the PBS control. The protein spots presenting differential level between cystamine and PBS treatment were then identified by peptide‐mass fingerprinting (PMF). After bioinformatic analysis, these identified proteins were found involved in mitochondrial apoptotic pathway, oxidative stress, and mitogen‐activated protein (MAP) kinase‐mediated pathway. Further investigation revealed that cystamine significantly decreased the levels of apoptotic Bax and Apaf‐1 and the activity of caspase‐3, and increased the levels of anti‐apoptotic Bcl‐2 in APM. We also found that these apoptotic mediators were up‐regulated in a correlation with the progression of lupus severity in NZB/W‐F1, which were little affected in BALB/c mice. We also found that the reduced serum glutathione was restored by cystamine in NZB/W‐F1. Interestingly, the phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2) in APM and the phagocytic ability was diminished in presence of cystamine. In conclusion, our findings indicate that cystamine significantly inhibited mitochondrial pathway, induced antioxidant proteins, and diminished phosphorylation of extracellular ERK1/2, which may alleviate the apoptosis and the phagocytic ability of APM. J. Cell. Biochem. 110: 660–670, 2010. © 2010 Wiley‐Liss, Inc.