Tissue Putrescine Research Articles

Influence of Some Heterocyclic, Cyclic, and Nitrogen-Containing Compounds on Oxidative Deamination of Polyamines in a Cell-Free Test System.

We studied the effects of some nitrogen-containing, heterocyclic, and cyclic compounds on the rate of oxidative deamination of polyamines and putrescine in tissues with a high proliferation rate. For this purpose, the specific activities of the main enzymes of polyamine oxidative degradation - spermine oxidase (SMO), polyamine oxidase (PAO), and diamine oxidase (DAO) were determined using a cell-free test system from regenerating rat liver. The compounds methyl 2-(5-formylfuran-2-yl)benzoate and 2,7-bis-[2-(diethylamino)ethoxy]-9H-fluoren-9-one (and in the form of dihydrochloride) showed mainly activating effect on oxidative degradation of putrescine, spermidine, and spermine, which indirectly indicates their antiproliferative effect. Nitrogen-free compounds inhibited this process, thus exhibiting potentially carcinogenic properties. Correlations were calculated for activity of DAO, PAO, and SMO with 5 topological indices: Wiener (W), Rouvray (R), Balaban (J) in the Trinaistich modification, detour (Ip), and electropy (Ie). The highest dependence was noted for DAO and the Balaban index (R=-0.55), for PAO and the detour index (R=0.78), and for SMO and the electropy index (R=0.53). The remaining dependencies showed insignificant correlation strength.

A simple, easy, and efficient HPTLC method for simultaneous determination of polyamines (putrescine, spermidine, and spermine) in plant tissues

Polyamine analysis has received a lot of attention in recent years because they are important in several physiological processes, including cell proliferation, differentiation, and growth. This study presents the development and validation of a user-friendly and convenient high-performance thin-layer chromatography (HPTLC) method, using benzoyl chloride as a derivatization agent for the analysis of three abundant polyamines. A comparative study with HPLC-MS/MS was performed to evaluate the putrescine, spermidine, and spermine content of plant tissues. The linear ranges for the determination of putrescine and spermine by HPTLC were lower than those of HPLC-MS/MS. Lower LODs were found for polyamine analysis using HPLC-MS/MS except for spermidine. The Pearson correlation coefficients of the two methods for putrescine, spermidine, and spermine were 0.982, 0.998, and 0.980, respectively, which confirms the applicability of both HPTLC-UV and LC-MS/MS methods. According to these results, the optimized HPTLC-UV method may be used as a practical analytical tool for screening polyamine levels in the horticultural, food, and pharmaceutical industries for quality control purposes.

Polyamines dynamics in buds and flowers in response to fall foliar fertilisation in ‘Hayward’ kiwifruit

ABSTRACT The present study aimed to optimise a fertiliser combination and find the effective timing for foliar fertilisation in the fall to determine whether the application of N, B and Zn fertilisers can improve fruit set and alter endogenous polyamines. ‘Hayward’ Kiwi vines were sprayed with CO(NH2)2 (urea), H3BO3 and ZnSO4 (alone and in combination) on different dates (September 17, October 7 and October 28). Buds were sampled at three stages (before dormancy, during dormancy and full bloom) to determine endogenous polyamines content. In the following growing season, bud break, percentage of flowering shoots and effective pollination period (EPP) were determined. Our results showed CO(NH2)2: H3BO3: ZnSO4 (10, 1.5 and 2 g l−1 respectively) on October 28 was the best fertiliser combination and application time. This treatment significantly increased putrescine, spermine and spermidine accumulation in bud and flower tissues, as well as bud break and flowering. In vines treated with this combination on October 28, EPP was at least 2 days longer than control. The foliar fertiliser treatment applied the previous fall increased fruit set to 83% (absolute). This improvement was associated with an increased accumulation of endogenous polyamines in bud and flower tissues.

Phase IIa Clinical Biomarker Trial of Dietary Arginine Restriction and Aspirin in Colorectal Cancer Patients.

After potentially curative treatment, colorectal cancer (CRC) patients remain at high risk for recurrence, second primary CRC, and high-risk adenomas. In combination with existing data, our previous findings provide a rationale for reducing tissue polyamines as tertiary prevention in non-metastatic CRC patients. The goal of this study was to demonstrate rectal tissue polyamine reduction in optimally treated stage I-III CRC patients after intervention with daily oral aspirin + dietary arginine restriction. A single-institution phase IIa clinical trial was conducted. Patients were treated with aspirin 325 mg/day and an individualized dietary regimen designed to reduce arginine intake by ≥30% over a 12-week study period. Dietary intake, endoscopy with rectal biopsies, and phlebotomy were performed pre- and post-intervention. The primary endpoint was to demonstrate ≥50% decrease in rectal tissue putrescine levels from baseline as a measure of polyamine reduction in the target tissue. Twenty eligible patients completed the study. After study intervention, mean dietary arginine intake decreased from 3.7 g/day ± 1.3 SD to 2.6 g/day ± 1.2 SD (29.7% decrease, p < 0.02 by Sign test). Mean plasma arginine levels decreased from 46.0 ng/mL ± 31.5 SD at baseline to 35 ng/mL ± 21.7 SD (p < 0.001). Rectal tissue putrescine levels were 0.90 nMol/mg-protein pre-intervention and 0.99 nMol/mg-protein post-intervention (p < 0.64, NS). No significant differences were observed for the other tissue polyamines investigated: spermidine (p < 0.13), spermine (p < 0.21), spermidine:spermine ratio (p < 0.71). Among CRC survivors, treatment with daily oral aspirin and an individualized dietary arginine restriction intervention resulted in lower calculated dietary arginine intake and plasma arginine levels but did not affect rectal tissue polyamine levels.

Systemic L-ornithine supplementation specifically increases ovarian putrescine levels during ovulation in mice†.

In all mammalian species examined thus far, the ovaries produce a burst of ornithine decarboxylase (ODC) and putrescine during ovulation or after application of human chorionic gonadotropin (hCG). Aged mice have significantly reduced levels of this periovulatory ODC and putrescine rise. Putrescine supplementation, in vitro during oocyte maturation or in mouse drinking water during the periovulatory period, reduces egg aneuploidies and embryo resorption, improving fertility of aged mice. These studies suggest that periovulatory putrescine supplementation may be a simple and effective therapy for reproductive aging for women. However, putrescine supplementation is expected to increase widespread tissue putrescine levels, raising concerns of nonspecific and unwanted side effects. Given that ODC is highly expressed in the ovaries during ovulation but otherwise exhibits low activity in most tissues, we hypothesized that periovulatory supplementation of L-ornithine, the substrate of ODC, might be suitable for delivering putrescine specifically to the ovaries. In this study, we have demonstrated that systemic application of L-ornithine via oral gavage or subcutaneous injection increased ovarian putrescine levels; the increase was restricted to animals that had been injected with hCG. Furthermore, L-ornithine specifically increased ovarian putrescine levels without affecting putrescine levels in any other tissues. However, our attempts to improve fertility of aged mice through L-ornithine supplementation in mouse drinking water produced either no effects (1% L-ornithine) or negative impact on fertility (4% ornithine). Our results suggest that it might not be feasible to achieve fertility-enhancing ovarian putrescine levels via L-ornithine supplementation in drinking water without encountering undesired consequences of high dose of exogenous L-ornithine.

Liquid Chromatographic Determination of Biogenic Amines in Fish Based on Pyrene Sulfonyl Chloride Pre-Column Derivatization

Monitoring of biogenic amines in food is important for quality control, in terms of freshness evaluation and even more for food safety. A novel and cost-effective method was developed and validated for the determination of the main biogenic amines: histamine, putrescine, cadaverine, spermidine and spermine in fish tissues. The method includes extraction of amines with perchloric acid, pre-column derivatization with Pyrene Sulfonyl Chloride (PSCl), extraction of derivatives with toluene, back-dissolution in ACN after evaporation and determination by reversed phase high performance liquid chromatography with UV and intramolecular excimer fluorescence detection. The structure of the pyrene-derivatives was confirmed by liquid chromatography–mass spectrometry with electrospray ionization. The standard addition technique was applied for the quantitation due to significant matrix effect, while the use of 1,7-diaminoheptane as internal standard offered an additional confirmation tool for the identification of the analytes. Method repeatability expressed as %RSD ranged between 7.4–14% for the different amines and recovery ranged from 67% for histamine up to 114% for spermine. The limits of detection ranged between 0.1–1.4 mg kg−1 and the limits of quantification between 0.3–4.2 mg kg−1. The method was applied to canned fish samples and the concentrations of the individual biogenic amines were below the detection limit up to 40.1 mg kg−1, while their sum was within the range 4.1–49.6 mg kg−1.

Maternal L-proline supplementation during gestation alters amino acid and polyamine metabolism in the first generation female offspring of C57BL/6J mice.

We recently reported that dietary supplementation with L-proline (proline) during gestation improved embryonic survival in C57BL/6J mice. The objective of the present study was to test the hypothesis that the effect of maternal proline supplementation on embryonic survival can be carried forward to the first generation female offspring. In the F0 generation, pregnant dams were fed a purified diet supplemented with 0 (control) or 5gproline/kg diet. The F1 female adult offsprings were bred to fertile males. Fetal survival at embryonic day (E)12.5 and reproductive outcomes at term birth were recorded. The concentrations of amino acids, ammonia, and urea in plasma and amniotic fluid, as well as concentrations of polyamines in placental tissues and amniotic fluid at E12.5 were determined. Results showed that the F1 generation female offspring from proline-supplemented dams had higher (P < 0.05) concentrations of glutamate and taurine in plasma; of putrescine and spermidine in placental tissues; and of glycine, taurine, and spermidine in amniotic fluid at E12.5, as compared with F1 generation female offsprings from dams without proline supplementation. Concentration of proline in the plasma of offspring mice from proline-supplemented dams were lower (P < 0.05), as compared with the control group. No differences in fetal survival, reproductive outcomes, or concentrations of ammonia and urea in plasma and amniotic fluid were observed between the two groups of F1 female offspring. Collectively, our results indicate that the benefits of maternal proline supplementation during gestation on improving embryonic survival and fetal growth in F0 females are not transmitted to their F1 generation females.

Restoration of Polyamine Metabolic Patterns in In Vivo and In Vitro Model of Ischemic Stroke following Human Mesenchymal Stem Cell Treatment.

We investigated changes in PA levels by the treatment of human bone-marrow-derived mesenchymal stem cells (hBM-MSCs) in ischemic stroke in rat brain model and in cultured neuronal SH-SY5Y cells exposed to oxygen-glucose deprivation (OGD). In ischemic rat model, transient middle cerebral artery occlusion (MCAo) was performed for 2 h, followed by intravenous transplantation of hBM-MSCs or phosphate-buffered saline (PBS) the day following MCAo. Metabolic profiling analysis of PAs was examined in brains from three groups: control rats, PBS-treated MCAo rats (MCAo), and hBM-MSCs-treated MCAo rats (MCAo + hBM-MSCs). In ischemic cell model, SH-SY5Y cells were exposed to OGD for 24 h, treated with hBM-MSCs (OGD + hBM-MSCs) prior to continued aerobic incubation, and then samples were collected after coculture for 72 h. In the in vivo MCAo ischemic model, levels of some PAs in brain samples of the MCAo and MCAo + hBM-MSCs groups were significantly different from those of the control group. In particular, putrescine, cadaverine, and spermidine in brain tissues of the MCAo + hBM-MSCs group were significantly reduced in comparison to those in the MCAo group. In the in vitro OGD system, N 1-acetylspermidine, spermidine, N 1-acetylspermine, and spermine in cells of the OGD + hBM-MSCs group were significantly reduced compared to those of OGD group.

Tissue and plasma putrescine levels in non-survivors of sepsis in a fluid-resuscitated rat model of faecal peritonitis

The polyamine, putrescine, was first isolated from putrefying meat but is thought to play an important role in cell growth and differentiation ([1]). It also generates succinate via GABA and can thus serve as an energy source to the small intestine ([2]). Elevated plasma putrescine levels have been reported in an endotoxic rodent model ([3]). We have previously characterized a 72 h fluid-resuscitated rat model of faecal peritonitis where prognostication can be accurately made as early as 6 h post-insult ([4]).

Simultaneous determination of eight underivatised biogenic amines in fish by solid phase extraction and liquid chromatography–tandem mass spectrometry

Biogenic amines on fish tissue are formed as a result of bacterial contamination and spoilage during storage. A new method based on liquid chromatography (LC) and tandem mass spectrometry (MS/MS) using a triple quadrupole (QqQ) analyser was developed for the analysis of eight biogenic amines (cadaverine, histamine, phenylethylamine, putrescine, spermine, spermidine, tyramine and tryptamine) in fish tissues. Sample preparation was performed by extraction with trichloroacetic acid 5% and solid phase extraction clean up with STRATA X cartridge. The MS/MS method was validated and compared with a method based on the analysis of dansyl derivatives by LC and fluorescence detector (FD). MS/MS achieved higher sensitivity (from 0.02mgkg−1 for spermidine and phenylethylamine to 0.2mgkg−1 for spermine) when compared to FD (from 1mgkg−1 for putrescine and tyramine to 4mgkg−1 for histamine); MS/MS method showed higher precision too, with intraday relative standard deviations (RSDs) from 1% to 4% with respect to those obtained with FD method (from 3% to 17%).Recovery study was conducted at two different fortification levels and the average ranged from 71% to 93% for all of the studied compounds with RSDs lower than 18%. Matrix-matched standards were used to counteract matrix effect observed in MS/MS determination. The applicability of the method was demonstrated by the analysis of biogenic amines in fish obtained from commercials of Valencia.

A phase IIa clinical biomarker trial of aspirin and dietary arginine restriction in colorectal cancer patients.

TPS132 Background: Colorectal cancer (CRC) patients are at high risk for recurrence and for secondary CRC development. Our preliminary findings provide a rationale for reducing tissue polyamines as tertiary chemoprevention in local and locally-advanced CRC patients. We will attempt to demonstrate rectal tissue putrescine reduction in stage I-III, optimally-treated CRC patients after treatment with dietary arginine restriction + daily oral aspirin in a phase IIa clinical biomarker trial. Methods: 24 participants will be treated with aspirin and an individualized dietary regimen to reduce arginine intake by >30% during the 12-week intervention. Sample size calculations were based on the Binomial test for a single proportion, to yield 80% Power. Six patients will receive psychosocial telephone counseling (PTC). Endoscopy with serial biopsies, phlebotomy, and urine collection will be performed pre- and post-intervention to assess potential biomarkers. The primary endpoint is a > 50% decrease in rectal tissue putrescine levels from baseline, as a measure of polyamine reduction in the target tissue of CRC patients. Secondary aims include: to demonstrate significant alterations in secondary endpoint biomarkers relevant to polyamine metabolism in CRC patients, including rectal mucosa Odc1 and Ssat expression and urinary polyamine metabolites; to determine this intervention’s side-effect profile in a population of optimally-treated CRC patients; to determine the efficacy of accrual to this novel type of clinical trial involving dietary intervention and oral aspirin therapy among a group of potentially cured cancer patients for whom there is currently no recommended further therapy; to assess feasibility of PTC and collect pilot data regarding the influence of PTC on compliance with the dietary prescription and compliance with aspirin, and if PTC further influences healthy exercise, reduced fatigue and improved quality of life when compared to the non-counseled cohort. Hypothesis: Among optimally-treated stage I-III CRC patients, a 12-week intervention of daily aspirin and a low-meat, arginine-restricted diet will favorably alter polyamine-related tissue, serum, and urinary biomarker levels.

Polyamine transport is mediated by both endocytic and solute carrier transport mechanisms in the gastrointestinal tract.

The polyamines spermidine and spermine, and their precursor putrescine, are required for cell growth and cellular functions. The high levels of tissue polyamines are implicated in carcinogenesis. The major sources of exogenous polyamines are diet and intestinal luminal bacteria in gastrointestinal (GI) tissues. Both endocytic and solute carrier-dependent mechanisms have been described for polyamine uptake. Knocking down of caveolin-1 protein increased polyamine uptake in colon cancer-derived HCT116 cells. Dietary supplied putrescine was accumulated in GI tissues and liver in caveolin-1 knockout mice more than wild-type mice. Knocking out of nitric oxide synthase (NOS2), which has been implicated in the release of exogenous polyamines from internalized vesicles, abolished the accumulation of dietary putrescine in GI tissues. Under conditions of reduced endogenous tissue putrescine contents, caused by treatment with the polyamine synthesis inhibitor difluoromethylornithine (DFMO), small intestinal and colonic mucosal polyamine contents increased with dietary putrescine levels, even in mice lacking NOS2. Knocking down the solute carrier transporter SLC3A2 in HCT116-derived Hkh2 cells reduced the accumulation of exogenous putrescine and total polyamine contents in DFMO treated cells, relative to non-DFMO-treated cells. These data demonstrate that exogenous putrescine is transported into GI tissues by caveolin-1- and NOS2-dependent mechanisms, but that the solute carrier transporter SLC3A2 can function bidirectionally to import putrescine under conditions of low tissue polyamines.

Ornithine decarboxylase (Odc)-1 gene polymorphism effects on baseline tissue polyamine levels and adenoma recurrence in a randomized phase III adenoma prevention trial of DFMO + sulindac versus placebo

1502 Background: The Odc G315A single nucleotide polymorphism (SNP) affects Odc transcription and favorably modifies aspirin's effect on colorectal adenoma risk. Treatment with the polyamine-inhibitory regimen difluoromethylornithine (DFMO) and sulindac has been shown to markedly decrease adenoma recurrence compared to placebo. Here we investigate modifying effects of the Odc G315A SNP on baseline tissue polyamine content and polyp recurrence after treatment with DFMO + sulindac or placebo. Methods: Data from the randomized phase III DFMO (500mg daily) + sulindac (150mg daily) colorectal adenoma prevention trial were analyzed. Odc genotyping was performed on patient-derived genomic DNA samples using allele-specific TaqMan probes. Baseline rectal tissue polyamine content was determined via HPLC. Clinicopathologic data were compared after stratification by genotype. Fisher's exact test or χ2 tests for independence were used for comparisons of categorical variables. Two-tailed t-tests or Kruskal-Wallis nonparametric analysis of variance were used for numerical comparisons between groups. Results: Data were available for 122 of 375 study patients. Odc genotype distribution was 54% GG, 5% AA, 41% GA. Patients with any Odc A- allele (AA/GA) were similar in age, gender, race, and prior aspirin use across treatment arms, as were Odc GG patients. Odc GG vs AA/GA patients had significantly higher median baseline rectal tissue putrescine (0.52 vs 0.31 nmol/mg protein, P=0.046) and spermidine (2.10 vs 1.65 nmol/mg protein, P=0.011) content. Among Odc AA/GA patients, 7/26 patients (27%) had recurrent adenoma in the DFMO + sulindac arm vs 14/30 patients (47%) in the placebo arm (P=0.13, absolute risk difference=20%). Among Odc GG patients, 6/39 patients (15%) had recurrent adenoma in the DFMO + sulindac arm compared with 14/27 (52%) placebo patients (P=0.002, absolute risk difference=37%). Conclusions: DFMO + sulindac treatment lowers the incidence of recurrent adenomata among adenoma patients, with pronounced effects observed among Odc homozygous GG patients. This may be related to baseline tissue polyamine differences across these genetically-defined groups. No significant financial relationships to disclose.

Overexpression of spermidine/spermine N1-acetyltransferase elevates the threshold to pentylenetetrazol-induced seizure activity in transgenic mice

Activation of polyamine catabolism in transgenic mice through an overexpression of spermidine/spermine N 1-acetyltransferase (SSAT) results in a massive overaccumulation of the diamine putrescine in most tissues including brain. Putrescine pool in transgenic animals was strikingly expanded in every six brain regions analyzed at present. Pons (23-fold), cerebellum (37-fold), cerebrum (34-fold), and hippocampus (16-fold) showed the greatest increases in putrescine levels. Moreover, the molar ratio of putrescine to spermidine was increased in the different brain regions of the transgenic animals on an average of nearly 40-fold. Upon an exposure of the animals to pentylenetetrazol (PTZ) infusions, a compound known to induce epilepsy-like seizure activity, the SSAT transgenic mice showed significantly elevated seizure threshold to both clonic and tonic convulsions in comparison with their syngenic littermates. This difference, however, disappeared when the animals were treated with ifenprodil prior to PTZ infusions. The latter compound acts as an antagonist of N-methyl- d-aspartate receptor by binding to the polyamine site of the receptor. Overexpression of SSAT likewise appeared to protect the transgenic animals from PTZ-induced neuron loss in the hippocampus. As putrescine is known to serve as a precursor to γ-aminobutyric acid (GABA), we carried out 1H NMR analyses the results of which revealed that the levels of the inhibitory amino acid GABA and its excitatory counterpart glutamate were indistinguishable in syngenic and transgenic animals in all brain regions analyzed. The present results suggest that the frequently observed enhanced accumulation of putrescine in response to brain insults belongs to neuroprotective measures rather than being a cause of the subsequent injury.

The difference in the stimulation by putrescine of DNA synthesis using DNA polymerase extracts of normal rat liver or of tumour tissue or host liver from tumour-bearing rats.

Putrescine biosynthesis is elevated before DNA replication, and a stimulation of DNA synthesis by 20 mM putrescine has been found using an in vitro DNA synthesizing system. Furthermore, this stimulation of DNA synthesis by putrescine involves a particular factor (factor PA). This factor PA stimulates DNA polymerases alpha, beta, and gamma, and is present in nuclei and mitochondria but not in cytoplasm. Factor PA loses about 80% of its activity by heating at 45 degrees C for 15 min or by hydrolysis with 100 mg ml(-1) Enzygel trypsin. These properties indicate that factor PA is a protein. Its size is estimated to be about 2.1 S. DNA synthesis in nuclear and mitochondrial DNA polymerase extracts from tumour tissues and host livers of tumour-bearing rats are not stimulated by 20 mM putrescine. However, the addition of excess factor PA to DNA synthesizing systems using DNA polymerase extracts from proliferative tissues again results in a stimulation of DNA synthesis by exogenous putrescine. These findings indicate that the stimulatory effect of DNA synthesis in vitro by exogenous putrescine is controlled by the ratio between factor PA and endogenously synthesized putrescine in proliferative tissues or that sent by the bloodstream from proliferative tissues. These results suggest that a non-stimulatory effect of putrescine on DNA synthesis may be diagnostic in tumour-bearing patients.

Ornithine decarboxylase activity and its gene expression are increased in benign hyperplastic prostate.

Ornithine decarboxylase (ODC) is the first key enzyme in the polyamine biosynthesis pathway. Polyamine is believed to participate in cellular proliferation and differentiation. To study the relationship between ODC and the pathogenesis of benign prostatic hyperplasia (BPH), the polyamine levels, ODC activities, and expression of ODC mRNA in benign hyperplastic and normal human prostates were assayed. Polyamine contents and ODC activities in tissue extracts were determined by reverse-phase high-performance liquid chromatography and spectrophotometric procedures, respectively. The ODC mRNA levels were assayed by Northern blot analysis. The contents of putrescine, spermidine, and spermine in BPH tissues were 2.2, 3.4, and 6.0 times higher than those in normal tissues, respectively; the ODC activity of BPH tissue was about 3.2 times higher than in normal tissue; the expression level of ODC mRNA in the BPH tissues was greater than that of normal tissues. The findings imply that 1) the increased ODC activity and polyamine content in prostatic tissue may correlate with the pathogenesis of BPH, and 2) the high level of ODC activity is induced by the overexpression of ODC mRNA.

Elevated N1‐Acetylspermidine Levels in Gerbil and Rat Brains After CNS Injury

The polyamine system is very sensitive to different pathological states of the brain and is perturbed after CNS injury. The main modifications are significant increases in ornithine decarboxylase activity and an increase in tissue putrescine levels. Previously we have shown that the specific polyamine oxidase (PAO) inhibitor N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) reduced the tissue putrescine levels, edema, and infarct volume after transient focal cerebral ischemia in spontaneously hypertensive rats and traumatic brain injury of Sprague-Dawley rats. In the present study, N1-acetyl-spermidine accumulation was greater in injured brain regions compared with sham or contralateral regions following inhibition of PAO by MDL 72527. This indicates spermidine/spermine-N1-acetyltransferase (SSAT) activation after CNS injury. The observed increase in N1-acetylspermidine levels at 1 day after CNS trauma paralleled the decrease in putrescine levels after treatment with MDL 72527. This suggests that the increased putrescine formation at 1 day after CNS injury is mediated by the SSAT/PAO pathway, consistent with increased SSAT mRNA after transient ischemia.

Effects of Acute Ethanol Exposure on Polyamine and Gamma-Aminobutyric Acid Metabolism in the Regenerating Liver

Recently, it has been suggested that ethanol-induced inhibition of liver regeneration results from decreases in hepatic putrescine levels and/or increases in hepatic gamma-aminobutyric acid (GABA)ergic activity. Because putrescine can be metabolized by diamine (DAO) and monoamine (MAO) oxidases to GABA, we documented the effects of acute ethanol exposure on hepatic MAO or DAO activity following partial hepatectomy (PHx) in rats. We also documented the effects of ethanol on GABA transaminase (GABA-T), the enzyme responsible for GABA metabolism in the liver, and tissue putrescine and GABA levels. Adult, male Sprague–Dawley rats (200–250 g) were treated with either ethanol (3 g/kg) or equal volumes of saline by gastric gavage 1 h prior to a 70% PHx or sham surgery. Rats were then sacrificed ( n = 5–7/group) at various times (0–72 h) post-PHx. Enzymatic activity and putrescine/GABA levels were determined by standard isotopic techniques and high-performance liquid chromatography respectively. Hepatic DAO activities in ethanol-treated rats were transiently higher than in saline-treated controls (30% increases at 6 h, p < 0.05). Hepatic MAO and GABA-T activities in acute ethanol-treated rats were essentially identical to saline-treated controls. Although hepatic putrescine levels were similar in ethanol- and saline-treated rats, hepatic GABA levels were approximately three times higher in ethanol-treated rats at 12 and 24 h post-PHx ( p < 0.0001). In conclusion, the results of this study indicate that acute ethanol exposure has a limited effect on the enzymatic conversion of putrescine to GABA following partial hepatectomy in the liver. The results also indicate that increased GABAergic inhibition rather than decreased putrescine stimulation is more likely to play a role in ethanol-induced inhibition of hepatic regeneration.

Genotypic Variability of de novo Shoot Morphogenesis of Brassica oleracea in vitro in Response to Ethylene Inhibitors and Putrescine

Effects of ethylene inhibitors and exogenous putrescine on the shoot regeneration capacity of cultured hypocotyl explants from 18 cultivars of cabbage, cauliflower and broccoli were investigated. All cultivars were poorly regenerative on Murashige and Skoog's medium supplemented with 0.5–1 mg · L −1 α-naphthaleneacetic acid in combination with 1–4 mg · L −1 benzyladenine. Shoot regeneration of some cultivars was enhanced by addition of 10 μmol · L −1 AgNO 3 to the medium. The presence of 10 μmol · L −1 aminoethoxyvinylglycine (AVG) or 10 mmol · L −1 exogenous putrescine was also beneficial to regeneration, whereas combining chemical treatment with vacuum infiltration had little effect. Explants of all three cabbage cultivars tested (‹Booming Spring›, ‹Eastern Riches› and ‹Summer Autumn›), when grown on AgNO 3 medium, produced significantly more ethylene than control explants, but there was no difference in the content of free polyamines (putrescine, spermidine and spermine) in both tissues. On the contrary, explants grown in the presence of AVG emanated lower ethylene during the three-week culture period. Furthermore, these explants also showed a significantly higher free putrescine content up to 7–10 days of culture, although the difference in free spermidine and spermine between AVG-grown and control explants was negligible. The results support the hypothesis that ethylene produced by explants often inhibits shoot regeneration in vitro .

Influence of potassium nutrition and the nitrate/ammonium ratio on the putrescine and spermidine contents in banana vitroplants

Potassium (K) is required in high doses by the banana (Musa sp.) plant and interacts with other nutrient elements in which banana tissues are maintained under in vitro condition as a consequence modifications in the plant metabolism take place mainly in nitrogen (N) compounds, such as proteins, amino acids, and secondary compounds. When K is present in concentrations lower than that required, diamines such as putrescine and poliamines are formed. This metabolic disorder can also be correlated with the presence of different inorganic N forms, such as nitrate (NO3) and ammonium (NH4), and the ratios between both ions as well. In order to follow the physiological performance of the interrelationships, K/putrescine and of the NO3/NH4 ratio in the tissue of banana vitroplantlets, shoot apex of two banana cvs. Nanica and Prata Ana were maintained in modified MS medium in the presence of six different doses of K: 5, 10, 15, 20, 25, and 30 mM. After the period of tissue proliferation the cultures were transferred to rooting media containing the same different K doses. Dry matter, K, putrescine, and spermidine contents and their accumulation were determined in the shoots and roots of the vitroplantlets and in the shoot apex of the expiant donor cultivar as well as the corresponding values for the whole vitroplantlets calculated. The data were statistically analyzed. The contents and accumulations of putrescine and spermidine in banana tissues were enhanced as K concentration decreased in the medium: four times (0.19% of the dry matter) for cv. Nanica and eight times (0.25% of the dry matter) for cv. Prata Ana. This behavior was not only related to the K depletion but to the NO3/NH4 ratio as well.