Adrenomedullary ornithine decar☐ylase activity: Its use in biochemical mapping of the origins of the splanchnic nerve in the rat

The activity of ornithine decar☐ylase in the adrenal medulla of the rat can be induced transsynaptically by the repeated administration of apomorphine. Unilateral section of one to four ventral spinal cord roots from T4 to T12 partially prevents this effect. Interruption of the intercostal nerves (T7–T13), with preservation of the autonomic innervation of the adrenal medulla, does not produce any alteration in the response of the medullary ODC to APO in the operated side as compared to the intact side. Dorsal root section at T7–T10 leads to a small reduction, while section at T2–T4 has no effect at all. Thus, selective surgical interruption of spinal cord roots indicates that the bulk of splanchnic fibers mediating the transsynaptic induction of adrenomedullary ornithine decar☐ylase course in the ventral roots between T7 and T10. Dorsal rhizotomy demonstrates a modulatory role in this induction of afferent information to sympathoadrenal preganglionic neurons involved in innervation of the chromaffin cells.     

Intact epidermal cell assay for ornithine decarboxylase activity

Summary A procedure measuring the ornithine decarboxylase (ODC) activity and polyamine formation of intact neonatal mouse epidermal cells in culture has been developed and tested. Basal cells prepared from neonatal mouse epidermis were plated on round 15-mm Lux coverslips, placed in Costar 24 well culture clusters and grown at 32°C in M-199 + 13% fetal bovine serum. Before assay the cells were rendered permeable to ornithine ¹⁴C and ODC inhibitors using the buffer described by Berger et al. [3]. The slides, covered with adhering cell layers, were then placed in vials, covered with assay buffer and assayed intact for ODC activity. The ODC reaction was terminated by addition of citric acid to the buffer and the amount of ¹⁴CO₂ released was determined by scintillation counting of a center well filled with trapping agent. The baseline ODC activity of the intact cells was 500–1,000 pmol ¹⁴CO₂/mg protein/45 min. The validity of this ODC assay procedure using intact neonatal mouse keratinocytes was tested by use of three specific ODC inhibitors and by measuring the formation of polyamines from uniform labeled ornithine. The results indicated that authentic ODC activity was measured and preserved in this intact neonatal mouse epidermal cell assay. This technique holds promise for future studies of epidermal cell regulation of ODC and polyamine synthesis and studies of the multiple ornithine metabolites and conjugates formed, using a highly manipulable in vitro system.     

Early rapid and transient increase in ornithine decarboxylase activity within sympathetic neurons after axonal injury

The activity of ornithine decarboxylase (ODC) was examined in the rat superior cervical ganglion during development and after postganglionic axotomy. During development the enzyme activity in the ganglion was transiently elevated at 19 days gestation (2 days before birth) and then declined to low adult activities by 5 days after birth. After axotomy the enzyme activity in the ganglion was rapidly increased. As early as 2 h after the injury the activity was increased to 200% and by 10 h it attained a peak of 453% above control values. The activity then rapidly declined to a lower value of about 200% above control by 24 h, where it remained as long as 5 days and then returned to control values by 10 days postoperative. The early increase in ODC activity was demonstrated by inhibitor cytochemistry to occur within neuronal cell bodies. The ODC activity was also increased after preganglionic nerve transection (denervation) (275%), end organ removal (60%), and 6-hydroxydopamine treatment (75%). We conclude that increased ODC activity is one of the earliest events in the reaction of the nerve cell body to axonal injury, and that increased ODC activity is a common feature of drastic metabolic alterations in neurons of the superior cervical ganglion.     

Impaired development of cerebellar cortex in rats treated postnatally with alpha-difluoromethylornithine

alpha-Difluoromethylornithine specifically and irreversibly inhibits the enzyme ornithine decarboxylase. Ornithine decarboxylase catalyses the initial step in the synthesis of polyamines, which are thought to play an essential role in growth and development of mammalian tissues. The current study examined the effects of alpha-difluoromethylornithine on the ontogenic development of the rat cerebellar cortex. Animals injected daily with alpha-difluoromethylornithine on postnatal days 1-21 suffered a deficit in the number of granule cells and many of the remaining granule cells became trapped in the molecular layer during migration. Purkinje cells were also scattered throughout the molecular layer and their mean diameter was 38% smaller than in controls. In general, the cerebellar cortex of alpha-difluoromethylornithine-treated rats failed to progress much beyond the stage of development reached in control rats during the first postnatal week. These effects of alpha-difluoromethylornithine were already clearly visible at 10-15 days of age. The final size of the cerebellum as a whole and of individual folia was markedly subnormal. These data indicate that polyamines play an obligatory role in cerebellar neurogenesis and histogenesis.     

Response of mouse skin to tattooing: use of SKH-1 mice as a surrogate model for human tattooing

Tattooing is a popular cosmetic practice involving more than 45 million US citizens. Since the toxicology of tattoo inks and pigments used to formulate tattoo inks has not been reported, we studied the immunological impact of tattooing and determined recovery time from this trauma. SKH-1 hairless mice were tattooed using commercial tattoo inks or suspensions of titanium dioxide, cadmium sulfide, or iron oxide, and sacrificed at 0.5, 1, 3, 4, 7, or 14 days post-tattooing. Histological evaluation revealed dermal hemorrhage at 0.5 and 1 day. Acute inflammation and epidermal necrosis were initiated at 0.5 day decreasing in incidence by day 14. Dermal necrosis and epidermal hyperplasia were prominent by day 3, reducing in severity by day 14. Chronic active inflammation persisted in all tattooed mice from day 3 to 14 post-tattooing. Inguinal and axillary lymph nodes were pigmented, the inguinal being most reactive as evidenced by lymphoid hyperplasia and polymorphonuclear infiltration. Cutaneous nuclear protein concentrations of nuclear factor-kappa B were elevated between 0.5 and 4 days. Inflammatory and proliferative biomarkers, cyclooxygenase-1, cyclooxygenase-2, and ornithine decarboxylase protein levels were elevated between 0.5 and 4 days in the skin and decreased to control levels by day 14. Interleukin-1 beta and interleukin-10 were elevated in the lymph nodes but suppressed in the tattooed skin, with maximal suppression occurring between days 0.5 and 4. These data demonstrate that mice substantially recover from the tattooing insult by 14 days, leaving behind pigment in the dermis and the regional lymph nodes. The response seen in mice is similar to acute injury seen in humans, suggesting that the murine model might be a suitable surrogate for investigating the toxicological and phototoxicological properties of ingredients used in tattooing.     

大鼠心肌多胺代谢限速酶ODC、SSAT活性分析

目的建立大鼠心肌多胺代谢限速酶鸟氨酸脱羧酶(ODC)及精脒/精胺乙酰转移酶(SSAT)活性分析方法。方法以Langendorff离体灌流心肌为实验材料,制备心肌组织匀浆;分别以dl[114C]Ornithine及[114C]acetylCoenzymeA为底物,以液体闪烁计数仪记录生成的14CO2及[14C]acetylspermidine的放射活度,并以其代表ODC,SSAT的活性;计算大鼠心肌ODC、SSAT的酶促反应动力学参数,筛选出适宜的底物浓度;同时观察一氧化氮(NO)供体硝普钠(SNP)对酶活性的影响。结果①大鼠心肌ODC、SSAT基础活性分别为:(9.67±3.09)nmol·mg-1Pro·h-1;(3.59±0.91)nmol·mg-1Pro·min-1。②ODC催化LOrnithine的酶促反应动力学参数Km=(54.95±8.14)μmol·L-1;Vmax=(2.364±0.37)nmol·mg-1·h-1;SSAT催化AcetylCoenzymeA的酶促反应动力学参数Km=(12.87±1.88)μmol·L-1;Vmax=(0.50±0.07)nmol·mg-1·min-1。③大鼠心肌ODC、SSAT活性检测的底物浓度分别为:90μmol·L-1(18.5kBq)DL[114C]Ornithine及36μmol·L-1(2.96kBq)[114C]acetylCoA。④SNP呈浓度依赖性地抑制ODC的活性、诱导SSAT的活性。结论建立了大鼠心肌多胺代谢限速酶鸟氨酸脱羧酶(ODC)及精脒/精胺乙酰转移酶(SSAT)活性的分析方法,该方法简便易行;根据Km值确定测定大鼠心肌ODC及SSAT?     

Garlic oil ameliorates ferric nitrilotriacetate (Fe-NTA)-induced damage and tumor promotion: implications for cancer prevention.

Intraperitoneal injection of ferric nitrilotriacetate (Fe-NTA) to rats and mice results in iron-induced free radical injury and cancer in kidneys. This study was designed to investigate the effects of garlic oil on Fe-NTA-induced damage and tumor promotion. Pretreatment of rats with garlic oil at a dose regimen of 50-100 mg/kg body weight for a week significantly and dose dependently protected against Fe-NTA induced damage as well as tumor promotion. Garlic oil afforded protection against hepatic lipid peroxidation, generation of hydrogen peroxide, preserved glutathione levels and activities of antioxidant enzymes. A protection against Fe-NTA induced hepatic tumor promotion was also apparent as inhibition in the modulation of hepatic tumor markers viz., ornithine decarboxylase activity and DNA synthesis. These results clearly demonstrate the role of oxidative stress and its relation to tumor promotion and suggest protective effects of garlic oil against Fe-NTA induced hepatic toxicity and it can serve as potent chemopreventive agent to suppress oxidant-induced tissue injury and carcinogenesis.     

A comparison of thyroxine- and polyamine-mediated enhancement of rat facial nerve regeneration

Thyroid hormones and spermidine, a motor neuron trophic polyamine (PA), have been shown to enhance peripheral motor nerve regeneration; however, the mechanism by which these treatment modalities exert their effect is unknown. Similarities in treatment outcome suggest that these molecules may be working via a common mechanism. Such an explanation is plausible since thyroid hormone is a potent inducer of ornithine decarboxylase (ODC), which is the rate-limiting enzyme involved in polyamine synthesis. This study was designed to morphologically evaluate the effects of exogenous thyroxine and spermidine on the regeneration of the rat facial nerve. Myelinated fiber density, axonal size, and degree of myelination were assayed by light and electron microscopy 21 days following facial nerve crush. Strikingly, the two treatment modalities had identical effects on all parameters tested. Each significantly enhanced the density of myelinated axons in regenerating nerves relative to the vehicle control. In addition, relative to the control treatment, both thyroxine and spermidine significantly increased the cross-sectional area of regenerating axons (P < 0.05). Interestingly, neither of the drug treatments had any effect on remyelination at the position where this parameter was analyzed. The concurrent administration of both thyroxine and spermidine did not synergistically enhance motor neuron regeneration. These data support the hypothesis that thyroxine and spermidine enhance neural regeneration by a common mechanism.