Puerarin Attenuates Cycloheximide-Induced Oxidative Damage and Memory-Consolidation Impairment in Rats.

BACKGROUND: Cycloheximide (CXM), an antifungal antibiotic, causes impaired memory consolidation as a side effect partially by disturbing the activities of the central catecholaminergic and cholinergic system. Some reports indicated that puerarin prevented memory impairment in various models in rodents. However, the protective effects of puerarin on the side effects of cycloheximide for memory consolidation impairment have not yet been investigated. METHODS: The protective effects of puerarin on CXM-induced memory-consolidation impairment, and memory impairment produced by central administration of AF64A neurotoxin, were investigated using a passive avoidance task in rats. A combination of transmitter receptor agonists and antagonists was used to explore the effects of puerarin on nervous system function. The activity of antioxidant defense systems and neurotransmitter systems in the prefrontal cortex and hippocampus were assayed. RESULTS: Systemic (25 and 50 mg/kg, i.p.) or central (5 and 10 µg/brain, i.c.v.) administration of puerarin attenuated CXM-induced memory-consolidation impairment produced by 1.5 mg/kg CXM (s.c.) in rats. The improvements produced by 50 mg/kg puerarin were blocked by cholinergic antagonists, a 5-HT2 receptor agonist, and an adrenergic receptor antagonist. Puerarin (only at 50 mg/kg, i.p.) reversed the CXM-induced alterations of the levels of norepinephrine in the prefrontal cortex and the levels of monoamines in the hippocampus. Puerarin also increased antioxidant-defense-system activities in the prefrontal cortex and hippocampus, which had been decreased by CXM. CONCLUSIONS: We suggested that the attenuating effects of puerarin on CXM-induced memory-consolidation impairment may be due to decrease oxidative damage and the normalition of the neurotransmitter function in the prefrontal cortex and hippocampus.

Continuing Impacts of Selective Inhibition on Bacterial and Fungal Communities in an Agricultural Soil.

Selective inhibition (SI) has been routinely used to differentiate the contributions of bacteria and fungi to soil ecological processes. SI experiments typically measured rapid responses within hours since the addition of inhibitor, but the long-term effects of selective biocides on microbial community composition and function were largely unknown. In this study, a microcosm experiment was performed with an agricultural soil to explore the effectiveness of two bactericides (bronopol, streptomycin) and two fungicides (cycloheximide, captan), which were applied at two different concentrations (2 and 10 mg g-1). The microcosms were incubated for 6 weeks. A radiolabeled substrate, [1,2,3,4,4a,9a-14C] anthracene, was spiked to all microcosms, and the derived CO2 was monitored during the incubation. The abundance and composition of bacteria and fungi were assessed by qPCR and Miseq sequencing of ribosomal rRNA genes. It was demonstrated that only 2 mg g-1 bronopol and cycloheximide significantly changed the bacteria to fungi ratio without apparent non-target inhibition on the abundances; however, community shifts were observed in all treatments after 6 weeks incubation. The enrichment of specific taxa implicated a selection of resistant or adapted microbes by these biocides. Mineralization of anthracene was continuingly suppressed in all SI microcosms, which may result in biased estimate of bacterial and fungal contributions to pollutant degradation. These findings highlight the risks of long-term application of selective inhibition, and a preliminary assessment of biocide selection and concentration is highly recommended.

TRIM11 cooperates with HSF1 to suppress the anti-tumor effect of proteotoxic stress drugs.

Cells mainly rely on stress proteins, such as heat-shock proteins (HSPs), to respond to various proteotoxic conditions. These proteins protect tumor cells and enhance their survive. However, the regulation of stress proteins involved in protein quality control (PQC) is still poorly understood. Here, we report that the expression of TRIM11, an important regulator of PQC, is positively correlated with tumor cell surviaval during the proteotoxic conditions induced by anti-tumor drugs. In addition, HSF1 is required for TRIM11-mediated removal of protein aggregates and resistance of proteotoxic stress. During these processes, TRIM11 interacts with and stabilizes HSF1, increaseing HSF1 levels in the nucleus. These findings identify that TRIM11, through cooperation with HSF1, protects cells against the proteotoxic stress and promotes tumor cell survival.

Low-level laser therapy prevents endothelial cells from TNF-α/cycloheximide-induced apoptosis.

Low-level laser therapy (LLLT), widely used in physiotherapy, has been known to enhance wound healing and stimulate cell proliferation, including fibroblast and endothelial cells. Applying LLLT can increase cell proliferation in many kinds of cells including fibroblasts and endothelial cells. However, the protective mechanisms of LLLT on endothelial apoptosis remain unclear. We hypothesized LLLT can protect endothelial cells from inflammation-induced apoptosis. Human endothelial cell line, EA.hy926 cells, and TNF-α/cycloheximide (TNF/CHX) were used to explore the protective effects of LLLT (660 nm) on inflammation-induced endothelial apoptosis. Cell viability, apoptosis, caspase-3/7/8/9 activity, MAPKs signaling, NF-κB activity, and inducible/endothelial nitric oxide synthase (iNOS/eNOS) expression were measured. Our results showed that LLLT increased EA.hy926 cell proliferation, attenuated the TNF/CHX-induced apoptosis, and reduced the TNF/CHX-mediated caspase-3/7/8/9 activation. In addition, LLLT increased ERK MAPK phosphorylation and suppressed the TNF/CHX-increased p38 MAPK, JNK, IKK phosphorylation, NF-κB translocation, and iNOS expression. The caspases-3 cleavage and cell death were not increased in cells treating with ERK inhibitor U0126, which implicated that ERK is not to be responsible for the protective effects of LLLT. After treating with p38 mitogen-activated protein kinase (MAPK) activator, the protection of LLLT in cell apoptosis was no longer existed, showing that LLLT protected the endothelial cells by suppressing p38 MAPK signaling. Our results provide a new insight into the possible molecular mechanisms in which LLLT protects against inflammatory-induced endothelial dysfunction.

6-Dimethylaminopurine and cyclohexamide are mutagenic and alter reproductive performance and intrauterine development in vivo.

The compounds 6-dimethylaminopurine (6-DMAP) and cyclohexamide (CHX) are currently used to stimulate the development of embryos produced by nuclear transfer in the production of cloned mammals with a great deal success. This study investigated the effects of 6-DMAP and CHX in vivo using biological assays to evaluate reproductive performance in females, teratogenesis, and mutagenesis. The results of this study demonstrated that the activating agents of oocyte cytoplasm, 6-DMAP and CHX, altered the reproductive performance of the experimental animals, as well as increased the rate malformations. In addition to these adverse effects, the administration of these compounds in pregnant females resulted in genotoxic and mutagenic toxicity, as determined by comet and micronucleus assays carried out in peripheral blood samples, respectively. Based on these findings and that alterations in DNA are important, morpho-functional teratogenesis and diminished embryonic viability, suggesting that 6-DMAP and CHX, which are utilized during the cloning of mammals, are responsible for the fact that embryos produced by nuclear transfer show low viability after implantation in utero or after birth because of congenital malformations and/or alterations in their DNA.

Reconsolidation of a long-term spatial memory is impaired by cycloheximide when reactivated with a contextual latent learning trial in male and female rats.

Reconsolidation of long-term memory has become a topic of great interest in recent years, and has the potential to provide important information regarding memory processes and the treatment of memory-related disorders. The present study examined the role of systemic protein synthesis inhibition in reconsolidation of a long-term spatial memory reactivated by a contextual latent learning trial in male and female rats. Using the Morris water maze, we demonstrate that: 1) a contextual latent reactivation treatment enhances memory, 2) systemic protein synthesis inhibition selectively impairs test performance when administered in conjunction with a memory reactivation treatment, and 3) that these effects are more pronounced in female rats. These findings indicate a role for protein synthesis in the reconsolidation of a contextually reactivated long-term spatial memory using the water maze, and a potential differential effect of sex in this apparatus. The role of the strength of the memory trace is discussed and the relevance of these findings to theories of reconsolidation and therapeutic treatment of post-traumatic stress disorder is discussed.

Cycloheximide impairs acquisition but not extinction of cocaine self-administration.

The aim of the present study was to assess the role of de novo protein synthesis in the acquisition and extinction of cocaine self-administration. In a first experiment, rats were trained to respond for intravenous cocaine infusions (0.3 mg/kg) and a protein synthesis inhibitor, cycloheximide (CHX; 3 mg/kg, s.c.) was injected immediately after each self-administration session. In a second experiment, rats were allowed to acquire cocaine self-administration and CHX was injected immediately after subsequent extinction sessions. CHX impaired the acquisition, but not extinction, of cocaine self-administration. In control experiments, CHX (3 mg/kg) blocked c-Fos protein expression after foot-shock stress and impaired the acquisition of conditioned freezing but did not inhibit spontaneous locomotor activity and sucrose drinking. Our results suggest that: i) the acquisition and extinction of cocaine-reinforced behaviour have a different molecular basis; and ii) only the former process requires de novo protein synthesis.

Toxicity and apoptosis induced by the mycotoxins nivalenol, deoxynivalenol and fumonisin B1 in a human erythroleukemia cell line.

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON) and fumonisin B1 (FB1) were studied in the K562 human erythroleukemia cell line using the Trypan Blue, MTT and BrdU uptake analyses of cytotoxicity, cell metabolism and cell proliferation, respectively. Nuclear staining with propidium iodide and DNA analysis by flow cytometry were used to identify apoptosis and cell cycle distribution. By the MTT and BrdU tests, both NIV and DON were significantly more toxic than FB1 by at least one order of magnitude, with ID50s ranging from 0.5 microM for NIV to 70 microM for FB1. The MTT test indicated that NIV was significantly (approximately four times) more toxic than DON. In contrast, the Trypan Blue test did not reveal any effects of mycotoxin exposure suggesting that, at the concentrations tested, NIV, DON and FB1 did not induce cytotoxicity through plasma membrane damage. Cell cycle analysis suggested apoptotic cytotoxicity, revealing 100% cellular debris at the highest concentrations of NIV and DON and approximately 2.9 times more debris than control at the highest FB1 concentration. Morphological evidence of apoptosis was related to the toxicity of the substances, such that the more toxic NIV and DON resulted in more late stage apoptotic events than FB1. This study suggests that human blood cells are sensitive to mycotoxin exposure, that NIV is more toxic than DON which is more toxic than FB1, and that DNA damage and apoptosis rather than plasma membrane damage and necrosis may be responsible for the observed cytotoxicity.

Differential impairment of auditory and contextual fear conditioning by protein synthesis inhibition in C57BL/6N mice.

A 1-trial fear conditioning was used to investigate the temporal development of fear responses expressed as increase of freezing or heart rate and its impairment by the protein synthesis inhibitor cycloheximide (CHX) in male C57BL/6N mice. Heart rate was measured with an implanted transmitter. In the memory tests, mice were exposed to tone and context provided either as foreground or background stimulus during training. The fear responses developed differently from 0 to 24 hr after training under these 3 conditions. A single pretraining CHX injection impaired both memory forms, whereas a single posttraining CHX injection impaired tone- but not context-dependent memory, with the context provided as background stimulus. It was concluded that consolidation of tone-, foreground context-, and background context-dependent fear conditioning may be mediated by partly different neuronal or partly different biochemical pathways, or both.

Antioxidants modulate induction of programmed endothelial cell death (apoptosis) by endotoxin.

OBJECTIVE: To evaluate the potential role of reactive oxygen metabolites as signals for endothelial cell apoptosis. DESIGN: A series of antioxidants were evaluated for their ability to block apoptosis in cultured porcine aortic endothelial cells in vitro. RESULTS: Scavenging of the hydroxyl radical with the membrane-permeable scavenger dimethyl sulfoxide or blocking its generation via the Fenton reaction by the chelation of iron with o-phenanthroline blocked apoptosis, whereas the cell membrane-impermeable scavengers superoxide dismutase and catalase did not block apoptosis. Inhibition of xanthine oxidase with enzyme-inhibitory levels of allopurinol also failed to block apoptosis, whereas high levels of allopurinol, which also scavenge the hydroxyl radical in vitro, conferred protection. In each case (dimethyl sulfoxide, o-phenanthroline, and high-dose allopurinol), hydroxyl radical ablation was only effective when administered before the priming step (lipopolysaccharide) and was ineffective when administered later, prior to the activation step (heat shock). CONCLUSIONS: These findings suggest a novel role for the hydroxyl radical as a nonlethal intracellular signal in endothelial cell apoptosis. Moreover, the results support a role for programmed cell death in the pathogenesis of multiple organ dysfunction syndrome and suggest novel strategies for prophylaxis and therapy of the most common cause of death in surgical intensive care units.

Studies on memory: spontaneous return of memory in 6-hydroxydopamine-treated mice and its relation to cycloheximide-induced transient amnesia.

We suggested previously that cycloheximide-induced transient amnesia was due in part to side-effects of the antibiotic on the central adrenergic system at the time of testing and that spontaneous return of memory depended upon recovery of the adrenergic system. To test these possibilities, mice chronically depleted of brain catecholamines (CAs) by 6=hydroxydopamine (6-OHDA) were trained in an avoidance-discrimination task and tested for spontaneous return of memory. Contrary to our hypothesis, amnesia at 24 hr after training was followed by recovery of memory by 72 hr, which indicates that recovery of memory can occur independently of adrenergic recovery. Injection of alpha-methyl-para-tyrosine immediately after training prevented return of memory at 72 hr, suggesting that the residual CAs remaining after 6-OHDA are necessary for memory to spontaneously return at this time.

Passive avoidance impairment in rats following cycloheximide injection into the amygdala.

Cycloheximide (CHX:1, 10 or 20 microg) was injected via indwelling cannulas into various regions of the rat brain and its effects on passive avoidance training were studied. Rats with 10 or 20 microg of CHX injected into the amygdala immediately after the training footshock exhibited amnesia for the learning experience when tested after 24 h. In contrast, animals injected with 20 microg of CHX at a site either in the internal capsule only 2 mm above the amygdaloid injection site or in the frontal cortex showed no retention deficit when tested after 24 h. A quantitative examination of protein synthesis in brain halves 30 min after unilateral injection of 20 microg of CHX into the amygdala demonstrated that total protein synthesis was inhibited by less than 10%. Autoradiographic studies revealed that this inhibition resulted from a profound, highly localized inhibition of protein synthesis in areas immediately adjacent to the cannula. A comparison of the regional patterns of protein synthesis inhibition caused by injection of CHX into either the amygdala or internal capsule suggested that CHX might produce amnesia by virtue of its localized effect on the amygdala. Control experiments revealed that injection of 20 microg CHX into the amygdala had no effect on short-term retention, or short-term performance. Injection of 20 microg of CHX into the amygdala 12 h after the footshock had no effect on long-term retention. The observed impairment of retention was shown to be dose-dependent as injection of 1 microg of CHX into the amygdala was without effect. In addition, it was demonstrated that the CHX-induced amnesia did not result from induction of local seizure activity. These data show that localized injections of small amounts of CHX into the amygdala can produce deficient memory of a training experience even though total brain protein synthesis is only slightly inhibited.

Some evidence for replication-transcription coupling in Physarum polycephalum.

Hydroxyurea, at concentrations of 40-60 mM, selectively and effectively blocked incorporation of thymidine into DNA. Inhibition occurred within 5-10 min of application of the agent when DNA synthesis was in progress, while the onset of replication at the beginning of S-phase and DNA synthesis in G2 phase were not affected. Uridine incorporation into TCA-precipitable material, in the presence of hydroxyurea, was significantly (up to 70%) inhibited in early S-phase of the cell cycle. Selective inhibition of RNA synthesis was confirmed for RNA separated into rRNA-rich and poly(A)-rich RNA fractions and analysed by the 2 kinds of DNA-RNA hybridization reactions. Uridine incorporation into poly (A) RNA was also inhibited under conditions where cycloheximide prevented maturation of nascent DNA molecules in early S-phase. We assume that chromatin which is replicating early DNA sequences may be a more competent template for transcription.