Early increases in transglutaminase activity and polyamine levels in a Mallory-Denk body mouse model

Rodents treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) are a model of two hepatic toxic manifestations: porphyria and the appearance of hepatic cytoplasmic protein aggregates (Mallory-Denk Bodies, MDBs). MDBs are induced after long-term DDC feeding, consist primarily of keratins 8 and 18, and contain glutamine-lysine cross-links generated by transglutaminases (TGs). TGs are Ca²⁺-dependent enzymes which catalyze the formation of covalent bonds between proteins and between proteins and polyamines. The aim of the current study was to investigate the time-course of TG hepatic activity in CF1 male mice either acutely or chronically treated with DDC and to correlate this activity with polyamine and porphyrin levels. On day 3 of the treatment, statistically significant increases in TG activity (75%), porphyrin content (6740%) and spermidine levels (73%) were observed. Although not statistically significant, at this time point putrescine levels showed an increase of 52%. The highest TG activity was observed on day 30 (522%), while porphyrin levels were still gradually increasing by day 45 (37,000%). From day 7 of the treatment and until the end of the experiment, putrescine levels remained increased (781%). Spermine levels were not affected by the treatment. The DDC-induced increases in putrescine and spermidine levels herein reported seem to be an early event contributing to the stimulation of liver TG activity, and thus to the promotion of cross-linking reactions between keratin proteins. This in turn would contribute to the formation of protein aggregates, which would lead to the appearance of MDBs. Due to the pro-oxidant and antioxidant properties of polyamines, it is possible to speculate that putrescine and spermidine may also participate at several levels in the oxidative stress processes associated with MDB formation.     

Spermine-Induced Alteration of Small Intestine in Suckling Rat: Involvement of Apoptosis or Zn2+ Enzymes?

Polyamines are of great importance in several physiological processes, such as cell proliferation and differentiation. The ingestion of spermine by suckling rats induces precocious maturation of their small intestine. Shortly after ingestion, spermine produces cell elimination at the villous top. The origin of this exfoliation was investigated to determine whether it was due to apoptosis. Wistar rats were orally treated with spermine. Apoptosis was analyzed in their small intestine by Tdt-mediated dUTP-fluorescein nick-end labeling reaction, caspase-3-like analysis, and DNA laddering. Polyamine content was measured by HPLC. The intestinal transitory alteration appeared as soon as 2 hr after spermine administration. Apoptosis events increased strongly at the same moment in the small intestine. They were evidenced by Tdt-mediated dUTP-fluorescein nick-end labeling analysis, DNA laddering, and caspase-3-like activity. Changes observed are consistent with apoptosis, but caspase inhibitor did not reduce intestinal alteration, as did Zn²⁺ chelator.     

Polyamines in prostatic epithelial cells and adenocarcinoma; the effects of androgen blockade.

BACKGROUND: Recent identification of eosinophilic prostatic secretory granules (PSG) as the major secretory mechanism of the prostate gland and their loss in neoplasia has prompted scrutiny of their chemical constituents. Polyamines, in particular spermine and spermidine (sp/spd) are the major cations found within prostatic secretions, yet their secretory mechanism in normal and neoplastic tissues has not been investigated. METHODS: Normal prostatic tissues and adenocarcinoma from intact and chemically castrated men were preserved in a glutaraldehyde-based fixative (Solufix((R))). Immunostains for sp/spd were performed before and after harsh acid hydrolysis whereby all protein was removed from tissue sections. RESULTS: Sp/spd immunoreactivity correlated with PSG as recognized in routine stains in tissues from intact patients before and after acid digestion. Decrease in sp/spd in untreated carcinomas was directly related to loss of PSG. After chemical castration, normal glands were mostly devoid of sp/spd while surviving malignant cells stained positively, despite a significant reduction or absence of PSG. Similarly, cancers progressing after castration were intensely decorated with anti-spermine, despite an almost complete loss of PSG. Cytoplasmic sp/spd staining of these androgen resistant clones was in contrast to normal glands no longer acid resistant. CONCLUSIONS: The intense eosinophilia of PSG is attributable to polyamines. Androgen blockade arrests sp/spd production in normal tissue. In contrast, sp/spd production continues in androgen resistant tumor clones, thereby uncoupling polyamines from their normal androgen dependent environment.     

Potent and long-lasting anticonvulsant effects of 1-naphthylacetyl spermine, an analogue of Joro spider toxin, against amygdaloid kindled seizures in rats

The anticonvulsant effect of 1-naphthylacetyl spermine (1-NA-Spm), an analogue of Joro spider toxin, against amygdaloid kindled seizures was studied in rats. 1-NA-Spm (10, 20 and 40 μg/rat) dose-dependently improved kindled seizures and shortened the afterdischarge duration 30 min after the administration. The anticonvulsant effect was observed even one day after the drug, and then gradually disappeared within 4 days. The present findings demonstrate that 1-NA-Spm acts as a potent and long-acting anticonvulsant against amygdaloid kindled seizures, and also suggest, together with the previous findings, that the calcium-permeable AMPA receptors, which are selectively antagonized by 1-NA-Spm, play a critical role in the seizure generation mechanism of amygdaloid kindling.     

Dietary Polyamines Intake and Risk of Colorectal Cancer: A Case-Control Study

Polyamines (including putrescine, spermidine, and spermine) are small, cationic molecules that are necessary for cell proliferation and differentiation. Few studies have examined the association of dietary polyamines intake with colorectal cancer risk. The aim of this study was to evaluate total polyamines, putrescine, spermidine, and spermine intake in relation to colorectal cancer risk in China. In total, 2502 colorectal cancer cases and 2538 age-(5-year interval) and sex-matched controls were recruited from July 2010 to April 2019. Odds ratios (ORs) and 95% confidence intervals (CI) were calculated by multivariable unconditional logistic regression after adjustment for various potential confounding factors. Higher intake of total polyamine, putrescine and spermidine was significantly associated with reduced risk of colorectal cancer. The adjusted ORs for the highest compared with the lowest quartile of intake were 0.60 (95% CI 0.50, 0.72; P_trend < 0.001) for total polyamines, 0.35 (95% CI 0.29, 0.43; P_trend < 0.001) for putrescine and 0.79 (95% CI 0.66, 0.95; P_trend = 0.001) for spermidine, respectively. However, higher intake of spermine was associated with increased risk of colorectal cancer, with an adjusted OR of 1.58 (95% CI 1.29, 1.93; P_trend < 0.001). This data indicate that higher intake of total polyamines, putrescine and spermidine, as well as lower intake of spermine, is associated with a decreased risk of colorectal cancer.     

Regulation of Ca2+ channel and phosphatase activities by polyamines in intestinal and vascular smooth muscle – implications for cellular growth and contractility

Polyamines added extracellularly to intestinal and vascular smooth muscle cells cause relaxation through inhibition of Ca²⁺ channel activity. Intracellularly applied polyamines also affect Ca²⁺ channel properties. Polyamines do not readily pass over the plasma membrane because of their positive charges but in permeabilized smooth muscle preparations they have free access to the cytoplasm. In this system they increase sensitivity of the contractile machinery to Ca²⁺ through inhibition of myosin phosphatase activity. The magnitude of Ca²⁺ channel and phosphatase inhibition depends on the number of positive charges on the polyamine molecule. Polyamines have an obligatory, but yet undefined, role in regulation of cell growth and proliferation. Several groups of protein kinases, such as tyrosine and mitogen activated protein (MAP)‐kinases transmit the growth signal from the plasma membrane to the cell nucleus where mitosis and protein synthesis are initiated. The data reviewed here show that polyamines may affect such signal transmission via inhibition of phosphatase activity.     

Behavioral and neurochemical effects of acute putrescine depletion by difluoromethylornithine in rats

It has been long known that the physiological concentrations of polyamines putrescine, spermidine and spermine are essential for cell growth. However, the role of endogenous polyamines in behavior function is poorly understood at present. This study investigated animals' behavioral performance and neurochemical changes in the hippocampus and prefrontal cortex following i.c.v. microinfusion of difluoromethylornithine (DFMO), a potent inhibitor of putrescine synthesis. Rats with low (25 μg) and high (50 μg) doses of DFMO spent significantly less time on the open arms and more time on the enclosed arms in the elevated plus maze relative to the saline controls, with no performance changes in the open field. The two DFMO groups were not impaired in the place and cued navigation, reversal training and probe tests in the water maze task. In the object recognition memory task, all three groups could detect the novel object, but rats in the high dose DFMO group spent significantly less time exploring displaced objects relative to the controls. DFMO treatment at both doses resulted in an 80%–90% reduction of putrescine level in the CA1, CA2/3 and dentate gyrus (DG) sub-regions of the hippocampus and the prefrontal cortex with minimal effects on the spermidine and spermine levels. Decreased spermidine/spermine molar ratio was found in DG and there were increased glutamate and GABA levels and their molar ratios in a region-specific manner in the DFMO groups. These results demonstrate that acute depletion of putrescine by DFMO produces anxiety-like behavior and impairs memory for the object displacement without affecting animals' locomotor and exploratory activity and spatial learning and memory. Multiple regression analysis data suggest the different roles of endogenous putrescine, spermidine and spermine on behavior function. The spermidine/spermine and glutamate/GABA ratios in hippocampal DG are strongly associated with anxiety-like behavior in the DFMO rats.     

The synthesis of deuterium‐labeled spermine,N1‐acetyl‐spermine and N1‐acetylspermidine

The synthesis of deuterium‐labeled spermine, N¹‐acetylspermine and N¹‐acetylspermidine is reported. 1,1,3,3‐²H₄‐N¹‐Acetylspermine hydrochloride, 1,1,3,3‐²H₄‐N¹‐acetylspermidine hydrochloride and 1,1,3,3,10,10,12,12‐²H₈‐spermine dihydrochloride were obtained in seven, four and three steps, respectively. All the syntheses were carried out by simple protection and deprotection steps from commonly used selective protecting reagents. These deuterium‐labeled compounds can be used as mechanistic probes of polyamine oxidizing enzymes. Copyright © 2007 John Wiley & Sons, Ltd.     

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?