Fever produced by intrahypothalamic pyrogen: effect of protein synthesis inhibition by anisomycin

In the unrestrained cat, the inhibition of protein synthesis by anisomycin, given either subcutaneously (5.0--25.0 mg/kg) or directly into the anterior hypothalamic, preoptic area (1.0--25.0 micrograms) impaired the development of a bacterial fever. S. typhosa infused intravenously (1:10 dilution in 1 ml) or into AH/POA (1.0 microliter) evoked an intense fever which was either significantly delayed or prevented by anisomycin. Conversely, anisomycin failed to affect the typical hyperthermia evoked by 100 ng PGE2 or 1.0--7.0 micrograms 5-HT similarly infused into AH/POA. These data demonstrate that an intermediary humoral factor of unknown nature is required in the hyperthermic effector pathway underlying the febrile response.     

Alleviation of anisomycin-induced amnesia by pre-test treatment with lysine-vasopressin

Amnesia in mice for a passive avoidance response induced by anisomycin injection immediately after training was reversed by 40 micrograms of lysine-vasopressin given one hour before testing. Control groups receiving non-contingent shock instead of training were used to demonstrate that the effects of vasopressin were due to memory of shock received in a particular place, rather than non-specific suppression of locomotion. The effects of vasopressin on retention were not mimicked by either pentylenetetrazol or epinephrine suggesting that the enhanced latencies were probably not the result of increases in fear or arousal. These data support the hypothesis that the retrieval of memory can be facilitated by vasopressin. The possibility of a relationship between the effects of vasopressin and those of catecholamine manipulations on memory is discussed.     

The effects of the protein synthesis inhibitor anisomycin on the febrile responses to intracerebroventricular injections of bacterial pyrogen,arachidonic acid and prostaglandin E2

Summary 1. Anisomycin (15 mg/kg) was administered s. c. to cats at ambient temperatures of 5°C, 20°C and 38°C. It produced biphasic effects on body temperature at 5°C and 20°C, an initial fall in temperature followed by a rise in body temperature, and a rise in body temperature of long latency at 38°C. 2. Anisomycin (15 mg/kg) attenuated the hyperthermic responses to centrally injected PGE₂ (1 g) at all ambient temperatures studied and also completely abolished the hyperthermic response to arachidonic acid (100 ng i. c. v.) at 20°C. 3. Shigella dysenteriae (100 ng i. c. v.) raised the body temperature of cats by increasing heat production and reducing heat loss at 5°C and 20°C, and by increasing heat conservation at 38°C. Anisomycin (15 mg/kg s. c.) pretreatment did not affect the temperature responses to the pyrogen at 20°C and 38°C, but did reduce the responses to Shigella dysenteriae (100 ng and 1 g i. c. v.) at 5°C. 4. Anisomycin (15 mg/kg s. c.) was administered to cats, 90 min after the injection of Shigella dysenteriae (100 ng i.e. v.), at 20°C at the onset of hyperthermia inn control experiments. Under these conditions, no hyperthermia was observed over a 2 h period following anisomycin injection. 5. It is concluded that anisomycin interferes with pyrogen induced fever by acting at a site after PGE₂ in the pathway to fever. Send offprint requests to: A. S. Milton     

Investigation of the reported protective effect of cycloheximide on memory

Many findings support the hypothesis that formation of long-term memory requires synthesis of proteins in the nervous system close to the time of learning. This hypothesis has been challenged recently by reports that the protein synthesis inhibitor cycloheximide (CYC) injected 2 hr prior to passive avoidance training in mice or rats attenuated the memory impairment induced by a usually amnestic dose of CYC administered 30 min pretraining. To investigate the reports of a "protective" effect of the prior injection, we attempted to replicate them and test their generality. For replication we administered either paired injections of CYC--120 mg/kg 2 hr prior to training and 30 mg/kg 30 min prior to training--or single injections of CYC (either 120 mg/kg or 30 mg/kg) 30 min pretraining and tested for retention of the passive avoidance habit either 1 or 7 days later. No attenuation of amnesia was observed at 1 day tests. Attenuation of amnesia following the double injection of CYC was observed at 7 day tests. When another protein synthesis inhibitor, anisomycin, was used in the same experimental design, there was no "protective" effect; two injections of anisomycin produced greater memory impairment for the passive avoidance habit than did the single low dose. Also, for active avoidance training, two successive injections of CYC caused significantly greater amnesia than did a single dose; this is the opposite of a "protective" effect. We suggest that the reported "protective" effect of CYC on memory is an as yet unexplained phenomenon that does not generalize to other antibiotic drugs and is specific to the passive avoidance task.     

茴香霉素对小鼠T细胞反应性及其杀伤作用的影响

目的 茴香霉素对小鼠T细胞活化、反应性、杀伤作用及其同种异基因皮肤移植的影响.方法 利用荧光标记的单克隆抗体双染技术结合流式细胞仪检测茴香霉素对小鼠CD3 T细胞早期及中期活化标志分子CD69和CD25表达的影响; 用MTT法检测茴香霉素刺激下T细胞在单向或双向混合淋巴细胞反应中T细胞的反应性及其对大鼠肝癌(7919)细胞的杀伤效应;建立小鼠同种异基因皮肤移植动物模型,观察茴香霉素对移植皮肤存活时间的影响.结果 在最佳剂量10.0 ng/mL时,茴香霉素能够明显抑制T细胞表面分子CD69和CD25的表达,且能抑制单向或双向混合淋巴细胞反应中T细胞的反应性(P＜0.01);10.0 ng/mL茴香霉素亦能明显抑制T细胞对大鼠肝癌细胞的杀伤效应(P＜0.01);5.0和15.0 mg/kg茴香霉素能够明显延长小鼠移植皮肤的存活时间 (P＜0.01).结论 茴香霉素对T 细胞的活化、反应性、杀伤效应及同种异基因皮肤移植排斥反应均有明显抑制作用,可能为治疗免疫反应性疾病提供新的策略 .     

Synergistic induction of TRAIL-mediated apoptosis by anisomycin in human hepatoma cells via the BH3-only protein Bid and c-Jun/AP-1 signaling pathway

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF super-family, and it has been shown that many human cancer cell lines are refractory to TRAIL-induced cell death. However, the molecular mechanisms underlying resistance are unclear. In the present study, we show that TRAIL-resistance is reversed in human hepatoma cells by anisomycin, which is known to inhibit protein synthesis and induce ribotoxic stress. Synergistic induction of apoptosis in cells treated with anisomycin plus TRAIL was associated with activation of caspases and cleavage of Bid, a pro-apoptotic BH3-only protein. Silencing of Bid expression by small interfering RNA (siRNA) significantly attenuated the loss of mitochondrial membrane potential (MMP, Δψ_m) and significantly increased induction of apoptosis in cells treated with anisomycin and TRAIL, confirming that Bid cleavage is required for the response. In addition, c-Jun/AP-1 was rapidly activated upon stimulation with anisomycin; however, the knockdown of c-Jun/AP-1 expression by c-Jun siRNA markedly reduced anisomycin plus TRAIL-induced loss of MMP and apoptosis. Taken together, the findings show that anisomycin sensitizes TRAIL-mediated hepatoma cell apoptosis via the mitochondria-associated pathway, involving the cleavage of Bid and activation of the c-Jun/AP-1 pathway, indicating that this compound can be used as an anti-tumor agent in combination with TRAIL.     

The role of protein synthesis in the hypothalamic mechanism mediating pyrogen fever

The effects of inhibition of protein synthesis by anisomycin on the pathogenesis of fever and normal thermoregulatory processes were investigated in the conscious and unrestrained cat. Subcutaneous administraton of 5.0–25.0 mg/kg of anisomycin prevented the fever normally evoked by an intravenous infusion of either 1.0 ml (10⁸ organisms) of a 1:10 dilution of S. typhosa or 1.0–5.0 ml (3.5×10⁵−2.1×10⁷ cells/ml) of endogenous pyrogen. In addition, systemic pre-treatment with anisomycin delayed and/or blocked the fever typically elicited by a direct micro-injection into the anterior hypothalamic, preoptic area (AH/POA) at AP 12.5–16.0 of 1.0 μl of the endotoxin. Anisomycin did not alter the hyperthermic response to an anterior hypothalamic injection of either 1.0–7.0 μg/1.0 μl of serotonin (5-HT) or 100.0 ng/1.0 μl of prostaglandin (PGE). Inhibition of protein synthesis, furthermore, did not prevent the fall in body temperature usually produced by an intrahypothalamic micro-injection of 2.33–14.0 μg/1.0 μl of either norepinephrine (NE) or dopamine (DA). The thermoregulatory capacity of the cat was unaffected by the administration of comparable doses of anisomycin, i.e., the animal was able to maintain normal body temperature (±0.5°C) when exposed to an ambient temperature of either 10°C or 34°C. These results strongly suggest that the synthesis of new protein within the region of the AH/POA is a functional requisite for the development of a pyrogen-induced fever.     

Effects of cycloheximide,a protein synthesis inhibitor,on mouse brain catecholamine biochemistry

Cycloheximide (CXM), a protein synthesis inhibitor, has been shown to result in a marked inhibition of central catecholamine (CA) synthetic mechanisms at doses that cause amnesia in animals. Unlike other inhibitors of CA synthesis no significant depletion of whole brain NE or DA concentrations was observed 0.75, 1, 2, 3, 4, 6, 17, or 24 hours after administration of CXM (120 mg/kg) to C57BL/6J mice. In order to investigate the underlying basis of maintenance of CA levels in face of CA synthesis inhibition, the effects of CXM on in vitro release of ³H-NE was studied in mouse hypothalamic slices. CXM, in a dose related manner, significantly inhibited the potassium stimulated release of NE from hypothalamic slices. Anisomycin, another protein synthesis inhibitor, similarly inhibited NE release. These studies further document the effects of protein synthesis inhibitors on CA mechanisms and suggest that disruption of CA biochemistry may play a role in the amnesia observed after administration of protein synthesis inhibitors.     

N-acetyltransferase and protein synthesis modulate melatonin production by Y79 human retinoblastoma cells

Melatonin is synthesized by the vertebrate pineal gland in a circadian fashion and is involved in numerous circadian and seasonal processes in the organism. The vertebrate retina also produces melatonin rhythmically to regulate rhythmic physiological processes in the eye. In both organs, melatonin is synthesized from serotonin by the sequential action of the enzymes, N-acethyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), and can be stimulated by increases in cyclic AMP through a mechanism requiring protein synthesis. The regulation of ocular melatonin biosynthesis in mammals and particularly humans, has not been well studied. Recently, we have shown that Y79 human retinoblastoma cells produce melatonin and that cAMP can stimulate melatonin production. Y79 cells, therefore, provide a model system in which to study melatonin synthesis in human tissue. We report that cAMP stimulates NAT, but not HIOMT activity in Y79 cells, and that stimulation of NAT activity is linearly related to melatonin release. In addition, the stimulation of NAT and melatonin requires protein synthesis. The turnover of NAT is rather rapid, with a half-life of about 20 min. These results suggest that the regulation of melatonin in Y79 retinoblastoma cells is similar to that found in the retina and pineal of other vertebrates.     

Effects of anisomycin and CNS stimulants on brain catecholamine synthesis.

Mice were injected with anisomycin, an inhibitor of cerebral protein synthesis; d-amphetamine, strychnine, or caffeine was administered 30 min later. Fifteen min before sacrifice at 1 or 2 hr after injection of anisomycin, 3H-tyrosine was injected intravenously, and catecholamine synthesis rates were estimated by measurement of the specific activity of 3H-tyrosine and the accumulation of 3H-norepinephrine and 3H-dopamine. Anisomycin decreased synthesis rates of catecholamines, but this effect was not significantly affected by any of the CNS stimulants. These results suggest that the recently reported reversal of anisomycin-induced amnesia by these stimulants is not due to an attenuation of brain catecholamine synthesis inhibition.