高效液相色谱测定大鼠早孕组织多胺的含量

建立大鼠早孕组织中多胺含量HPLC测定的方法。方法：大鼠早孕组织中多胺用10％三氯乙酸提取，苯甲酰氯柱前衍生，反相HPLC测定。结果：多胺中腐胺、精脒和精胺能较好分离；腐胺检测限可达1nmol、精脒和精胺可达5nmol；腐胺在1～1000nmol、精脒和精胺在5～1000nmol范围内线性良好；腐胺、精脒和精胺平均生物样本回收率分别为95．8％、92．8％和81．7％，日内变异系数（CV％）分别为2．46％、1．35％和1．94％，日间变异系数分别为3．49％、1．48％和3．27％。结论：该方法简便、灵敏，完全能满足大鼠早孕组织中多胺含量测定的需要。     

Effects of dietary polyamines at physiologic doses in early-weaned piglets

ObjectivePolyamines are essential for many cell functions, and they form part of the composition of maternal milk; despite this, their addition to infant formulas is currently under evaluation. The aim of the present study was to evaluate the effects of milk formulas designed to resemble sow milk supplemented with polyamines at maternal physiologic milk doses on the gut maturation of early-weaned piglets.MethodsWe fed 30 newborn piglets with maternal milk (n = 10), a control milk formula (n = 10), or a milk formula supplemented with polyamines (5 nmol/mL of spermine and 20 nmol/mL of spermidine, n = 10) for 13 d (day 2 after birth through day 15). Several growth and intestinal development parameters were measured.ResultsThe piglets fed the formula containing polyamine at physiologic doses showed significantly increased crypt depth in the small intestine compared with those fed with the control formula. Villus length was correlated to crypt depth. Although there were no differences in the disaccharidase activities between the animals fed the two formulas, alkaline phosphatase and γ-glutamyl transferase activities tended to be higher in the jejunum of those fed the polyamine-supplemented diet. Dietary polyamines did not significantly modify the gut mucosal concentrations of putrescine, spermine, or spermidine.ConclusionMilk formulas supplemented with polyamines at maternal milk physiologic doses slightly enhanced gut growth and maturation in neonatal piglets.     

Age and gender differences in response to neonatal ethanol withdrawal and polyamine challenge in organotypic hippocampal cultures

Background: Polyamines are synthesized and released in high concentrations during CNS development. These agents can potentiate N‐methyl‐D‐aspartate receptor (NMDAR) function and appear to play an important role in CNS development. Previous work has shown that polyamine release is increased during ethanol withdrawal (EWD). This likely promotes NMDAR overactivity and contributes to neurotoxicity during EWD, however, little is known regarding such effects in early neonatal brain. The present study compared the effects of EWD and polyamine exposure on toxicity in hippocampal slice cultures derived from postnatal day 2 (PND 2) or postnatal day 8 (PND 8) day‐old rats. Due to changes in NMDAR subtypes and response to polyamines, we predicted that slices taken from PND 2 pups would be more sensitive to EWD and polyamine challenge. Methods: Organotypic hippocampal slice cultures were obtained from neonatal rats either 2 or 8 days of age (PND 2 or PND 8). Five days after explantation, cultures were exposed to ETOH (50 mM‐ typically subthreshold for EWD induced cell death) for 10 days and then withdrawn from ETOH for 24‐hour in the presence of 100 μM of the polyamine spermidine and/or 100 μM ifenprodil, an NMDAR antagonist that blocks the NMDAR that is the most sensitive to polyamine modulation. Cytotoxicity was measured after 24‐hour by visualization of propidium iodide (PI) fluorescence. Results: There were clear age and gender‐dependent differences in response to EWD and to polyamines. EWD produced significant increases in PI uptake in all subregions (CA1, CA3 and DG) of cultures derived from PND 2 pups, but not PND 8 pups. Exposure of cultures to spermidine for 24‐hour also produced significant increases in cytotoxicity in all 3 regions of PND 2 cultures with no gender differences. In contrast, there were both gender and region‐specific differences in response to spermidine in cultures from PND 8. While the CA1 region of both sexes displayed increased cytotoxicity following spermidine exposure, only females showed increased cytotoxicity in the CA3 region while the DG appeared relatively insensitive to spermidine. Exposure to spermidine during EWD produced enhanced toxicity in all 3 hippocampal subregions in tissue from both PND 2 and PND 8 rats and this was reduced or prevented by co‐exposure to ifenprodil. Of interest, the PND 2 hippocampus was significantly more sensitive than the PND 8 hippocampus to the toxic effects of EWD and to spermidine during EWD in the DG and CA3 regions. Conclusions: Hippocampal slice cultures derived from PND 2 rats were more sensitive to the toxic effects of both EWD and EWD + spermidine exposure than were those derived from PND 8 rats. These findings are similar to recent behavioral data collected from our lab showing greater sensitivity to ETOH’s behavioral teratogenic effects when ETOH exposure in vivo occurred during the first postnatal week relative to the second postnatal week. Ifenprodil’s ability to block the toxic effects of spermidine during EWD suggests that excess activity of NR2B subunits of the NMDAR contributed to the excitatory and cytotoxic effects of EWD plus spermidine. While no sex differences in toxicity were observed in cultures taken from pups during the first postnatal week, these data do suggest that later in neonatal life (i.e., the second postnatal week), the female hippocampus may be more sensitive to polyamine‐induced neurotoxicity than males.     

Polyamine inhibition preserves somatosensory evoked potential activity after transient cerebral ischaemia

We tested the hypothesis that the increase in polyamines observed after cerebral ischaemia is related to deficits in electrocortical function as measured by somatosensory evoked potential (SEP). Adult Mongolian gerbils were anaesthetized with ketamine and prepared for monitoring SEP, cerebral blood flow (CBF) in parietal and frontal regions by H₂ clearance, and for bilateral carotid artery occlusion (BCO). Seven animals served as controls and received saline. Another 7 animals were treated with the ornithine decarboxylase inhibitor; difluoromethyl-ornithine (DFMO) (100 mg/kg I.P.) just prior to 40 min BCO followed by 4 h reperfusion. With BCO, both CBF and SEP declined significantly. In control animals, CBF fell from basal 37.8 ± 4.7 cc/100 g/min to 2.9 ± 1.2 cc/100 g/min and recovered to 22.7 ± 3.5 cc/100 g/h over the 4 h reperfusion period. DFMO treatment did not alter this CBF pattern. SEP amplitude declined to 11.3 ± 3.2% basal during occlusion. DFMO preserved SEP during ischaemia (35.5 ± 16.8% basal) and remained significantly more preserved during reperfusion (p > 0.05). These results suggest that polyamines are involved in the progressive decline in neuroelectrical function which occurs during occlusion/reperfusion in the Mongolian gerbil. The observation that polyamine inhibition preserves electrical function despite not altering blood flow indicates that the effects of polyamines are not manifested at the level of the vasculature but perhaps at the neuronal membrane.     

TGF-β1 inhibits polyamine biosynthesis in K 562 leukemic cells

Summary The present study proved that TGF-₁ significantly inhibited the growth of K 562 cells. The drop in cell numbers after 24 h incubation with increasing concentrations of TGF-₁ (0.01, 0.1, 1.0, 10.0 ng/ml) was accompanied by significant suppression of the activity of two key enzymes of polyamine biosynthesis: ornithine decarboxylase (ODC) and S-adenosylme-thionine decarboxylase (SAMDC). In contrast to ODC and SAMDC activity, TGF-₁ did not significantly affect the absolute concentration of spermidine and spermine in K 562 cells. We suppose that the lack of an evident drop in concentration of spermidine and spermine in spite of a significant decrease in ODC and SAMDC activity in K 562 cells exposed to TGF-₁ resulted from the uptake of polyamines from the extracellular space.     

Probiotics against neoplastic transformation of gastric mucosa: Effects on cell proliferation and polyamine metabolism

Gastric cancer is still the second leading cause of cancer death worldwide, accounting for about 10% of newly diagnosed neoplasms. In the last decades, an emerging role has been attributed to the relations between the intestinal microbiota and the onset of both gastrointestinal and non-gastrointestinal neoplasms. Thus, exogenous microbial administration of peculiar bacterial strains (probiotics) has been suggested as having a profound influence on multiple processes associated with a change in cancer risk. The internationally accepted definition of probiotics is live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. The possible effects on the gastrointestinal tract following probiotic administration have been investigated in vitro and in animal models, as well as in healthy volunteers and in patients suffering from different human gastrointestinal diseases. Although several evidences are available on the use of probiotics against the carcinogen Helicobacter pylori, little is still known about the potential cross-interactions among probiotics, the composition and quality of intestinal flora and the neoplastic transformation of gastric mucosa. In this connection, a significant role in cell proliferation is played by polyamines (putrescine, spermidine, and spermine). These small amines are required in both pre-neoplastic and neoplastic tissue to sustain the cell growth and the evidences here provided suggest that probiotics may act as antineoplastic agents in the stomach by affecting also the polyamine content and functions. This review will summarize data on the most widely recognized effects of probiotics against neoplastic transformation of gastric mucosa and in particular on their ability in modulating cell proliferation, paying attention to the polyamine metabolism.     

Putrescine produces antidepressant-like effects in the forced swimming test and in the tail suspension test in mice

Putrescine, a polyamine present at high concentrations in the mammalian brain, was suggested to play a role in the modulation of depression. Thus, this study investigated the effect of putrescine in the mouse forced swimming test (FST) and in the tail suspension test (TST), two models predictive of antidepressant activity. Putrescine significantly reduced the immobility time both in the FST and in the TST (dose range of 1–10 mg/kg, i.p.), without changing locomotion in an open-field. I.c.v. injection of putrescine (0.1–10 nmol/site) also reduced the immobility time in the FST and in the TST. The pretreatment of mice with arcaine (1 mg/kg, i.p., an antagonist of the polyamine-site of NMDA receptor) completely blocked the anti-immobility effect of putrescine (10 mg/kg, i.p.). A subeffective dose of putrescine (0.1 mg/kg, i.p.) produced a synergistic antidepressant-like effect with agmatine (0.001 mg/kg, i.p.) in the FST. Moreover, a subeffective dose of putrescine (0.01 nmol/site, i.c.v.) produced a synergistic antidepressant-like effect with arcaine (50 μg/site, i.c.v.). The results indicate that putrescine produces antidepressant-like effects in the FST that seems to be mediated through its interaction with the polyamine-site of NMDA receptors.     

A new enzymatic assay for total diamines and polyamines in urine of cancer patients

Summary A detailed procedure of a new photometric assay for total diamines and polyamines in human urine using soybean seedling amine oxidase (SSAO) as an enzyme reagent is described. It is based on the unique substrate specificity of SSAO that the enzyme is active toward all diamines and polyamines. The amines were purified from urine by cation-exchange chromatography and incubated with SSAO. Hydrogen peroxide formed in the oxidase reaction was measured photometrically by coupling 4-aminoantipyrine with phenol in the presence of peroxidase. For its simplicity and sensitivity, our method seems useful for routine clinical investigation. The data obtained from normal subjects and patients of various cancers are presented to validate the present method.Abbreviations SSAO soybean seedling amine oxidase - Put putrescine - Cad cadaverine - Spd spermidine - Spm sprmine     

Critical periods for the role of ornithine decarboxylase and the polyamines in growth and development of the rat: Effects of exposure to α-difluoromethylornithine during discrete prenatal or postnatal intervals

The roles of ornithine decarboxylase (ODC) and the polyamines in fetal and neonatal development were examined through the use of α-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC. Administration to pregnant rats of 500 mg/kg of DFMO every 12 h for a 4-day period (8 DFMO injections) resulted in fetal and neonatal death; DFMO early in gestation produced fetal resorption whereas late gestational exposure did not compromise fetal viability but instead resulted in a delayed toxic effect, with high mortality in the first postnatal week. Generalized toxicity of DFMO was not apparent in later developmental periods, as 4 days of DFMO treatment begun postnatally did not produce any neonatal death. Shortening the course of gestational DFMO treatment to 2.5 days (5 DFMO injections) also did not adversely affect fetal or neonatal viability and thus permitted identification of critical periods in which various tissues are sensitive to DFMO. Examination of growth patterns of brain, heart and kidney and of neurochemical development of central and peripheral catecholaminergic neurons indicated that different critical periods exist for effects of DFMO on each tissue or even on the various cell types within a tissue. The separable sensitivities were apparent even though the effects of DFMO on ODC and the polyamines for any given treatment period were fairly uniform in all tissues studied. These results indicate that the ODC/polyamine system plays multiple roles in fetal survival and in tissue growth during discrete periods of development; because the time course of cellular maturation differs for each tissue or cell population, DFMO administered during any one brief period can produce organ-specific developmental deficits.