Regulatory role of l-proline in fetal pig growth and intestinal epithelial cell proliferation

l-proline (Pro) is a precursor of ornithine, which is converted into polyamines via ornithine decarboxylase (ODC). Polyamines plays a key role in the proliferation of intestinal epithelial cells. The study investigated the effect of Pro on polyamine metabolism and cell proliferation on porcine enterocytes in vivo and in vitro. Twenty-four Huanjiang mini-pigs were randomly assigned into 1 of 3 groups and fed a basal diet that contained 0.77% alanine (Ala, iso-nitrogenous control), 1% Pro or 1% Pro + 0.0167% α-difluoromethylornithine (DFMO) from d 15 to 70 of gestation. The fetal body weight and number of fetuses per litter were determined, and the small and large intestines were obtained on d 70 ± 1.78 of gestation. The in vitro study was performed in intestinal porcine epithelial (IPEC-J2) cells cultured in Dulbecco''s modified Eagle medium-high glucose (DMEM-H) containing 0 μmol/L Pro, 400 μmol/L Pro, or 400 μmol/L Pro + 10 mmol/L DFMO for 4 d. The results showed that maternal dietary supplementation with 1% Pro increased fetal weight; the protein and DNA concentrations of the fetal small intestine; and mRNA levels for potassium voltage-gated channel, shaker-related subfamily, member 1 (Kv1.1) in the fetal small and large intestines (P < 0.05). Supplementing Pro to either gilts or IPEC-J2 cells increased ODC protein abundances and polyamine concentrations in the fetal intestines and IPEC-J2 cells (P < 0.05). In comparison with the Pro group, the combined administration of Pro and DFMO reduced the expression of ODC protein and spermine concentration in the fetal intestine, as well as the concentrations of putrescine, spermidine and spermine in IPEC-J2 cells (P < 0.05). Meanwhile, the percentage of cells in the S-phase and the mRNA levels of proto-oncogenes c-fos and c-myc were increased in response to Pro supplementation, whereas depletion of cellular polyamines with DFMO increased tumor protein p53 (p53) mRNA levels (P < 0.05). Taken together, dietary supplementation with Pro improved fetal pig growth and intestinal epithelial cell proliferation via enhancing polyamine synthesis.     

水稻丙酮酸脱羧酶基因OsPDC3功能的初步研究

 植物花粉中存在着活跃的有氧发酵过程。丙酮酸脱羧酶（PDC）作为发酵途径中的关键酶，参与花粉中的能量和物质代谢，在花粉萌发过程中起重要作用。通过反向遗传学的方法对水稻丙酮酸脱羧酶基因OsPDC3的功能进行了初步分析。OsPDC3是一个单外显子基因，编码蛋白与另外4个水稻PDC蛋白间的序列一致性达到77%~82%。表达模式分析显示OsPDC3在花粉中特异表达，GUS组织染色进一步证实其启动子具有花粉特异表达活性。超量表达OsPDC3会使转基因植株叶片中的PDC酶活性上升，表明OsPDC3在体内具有活性功能，能够参与花粉内的生理活动。反义抑制下调了OsPDC3在花粉中的表达水平，但对花粉的离体萌发率没有产生影响，推测这是由于PDC基因间的冗余造成的。     

Inhibition of mycelial growth and sporidial formation in the wheat pathogenNeovossia indica by a polyamine biosynthetic inhibitor

DL-α-Difluoromethylornithine (DFMO), a specific suicide inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase (EC 4.1.1.17), strongly inhibited mycelial growth and sporidial formation of the wheat pathogen,Neovossia indica, in vitro, while DL-α-difluoromethylarginine (DFMA), the analogous suicide inhibitor of arginine decarboxylase (EC 4.1.1.19), did not. The inhibited mycelial growth and sporidial formation were not only restored by putrescine (polyamine) addition, but were actually enhanced in the putrescine + DFMO cultures. Besides altering mycelial growth and morphology, DFMO also reduced the cell size drastically. The inhibition of fungal polyamine biosynthesis is discussed in relation to selective control of plant disease.     

Identification and quantification of amines in the equine caecum

Acute laminitis has been associated with the release of compounds, as yet unidentified, produced by hindgut fermentation which affect blood flow to the digit. The objectives of this study were to identify amine compounds in equine caecal and colonic contents, some of which are known to have vasoactive properties. In addition, the concentrations of amines in caecal contents of horses fed either grass or hay diets were compared. Fifteen amines were identified in equine hindgut contents in concentrations greater than 1 microM. The caecal concentrations of phenylethylamine, isoamylamine, cadaverine, diaminoheptane and spermidine were significantly higher in horses on spring/summer grass compared with those on winter grass or hay. These data show that many amines are present in the equine hindgut, some of which may have the potential to cause peripheral vasoconstriction if released into the circulation from the gastrointestinal tract.     

Enriched production of N-hydroxycinnamic acid amides and biogenic amines in pepper (Capsicum annuum) flowers

The biosynthesis of N-hydroxycinnamic acid amides (HCAAs), such as feruloyltyramine, p-coumaroyltyramine, feruloylserotonin, and p-coumaroylserotonin, in pepper plants was investigated in view of the activity of their corresponding enzymes and substrate levels. Almost all HCAAs were found in flowers, whereas the enzyme activity essential for HCAA biosynthesis seemed to be expressed constitutively in all tissues tested. The highest levels of the biogenic amines tyramine and serotonin, the key amine substrates, were also found in flowers, indirectly suggesting that the amine substrates play an important regulatory role in the synthesis of HCAAs.     

紫花苜蓿甘氨酸脱羧酶H-蛋白基因MsGDC-H1功能分析

光呼吸通过清除2-磷酸乙醇酸（2-PG）使氧合光合作用成为可能,该过程对C₃植物至关重要。H-蛋白是光呼吸过程中将甘氨酸转化为丝氨酸的甘氨酸脱羧酶（GDC）的关键组成蛋白之一。本研究克隆了紫花苜蓿MsGDC-H1,该基因编码166个氨基酸,具有1个硫辛酰基附着位点保守结构域和1个N6-硫辛酰赖氨酸保守位点。进化分析表明,MsGDC-H1蛋白与双子叶植物的甘氨酸脱羧酶H-蛋白（GDC-H）亲缘关系近。表达模式分析表明,MsGDC-H1在苜蓿叶中表达丰度高,且受光诱导。为了探究MsGDC-H1基因对拟南芥生长的影响,分别使用光诱导的茎叶特异性启动子ST-LS1和组成型启动子CaMV 35S驱动MsGDC-H1（ST-LS1：：MsGDC-H1; CaMV 35S：：MsGDC-H1）在拟南芥中异源表达。检测过表达植株生物量、淀粉、可溶性糖含量以及光合速率。数据分析显示,CaMV 35S：：MsGDC-H1过表达拟南芥（G系列植株）生长受阻,淀粉含量比ST-LS1：：MsGDC-H1特异性表达拟南芥（GS系列植株）增加了34%~67%,比野生型（WT）增加了7.3%~33.7%;可溶性糖含量比GS系列降低了36%~38%,比WT增加了44.3%~49.7%;与WT相比,GS系列植株生长更快,淀粉含量无显著差异（P>0.05）,可溶性糖含量显著增加（P<0.05）。试验结果表明,MsGDC-H1基因在调控拟南芥光合速率、碳水化合物合成以及生长等方面发挥重要作用,未来可作为提高苜蓿产量的基因工程育种候选基因。     

A comparison of RNA concentrations and ornithine decarboxylase activity in Atlantic salmon (Salmo salar) muscle tissue,with respect to specific growth rates and diel variations

RNA concentrations and enzyme activities are often used as indices of recent growth in fish, but few studies have used both methods to assess the same fish. This study measured RNA concentrations and ornithine decarboxylase (ODC) activity in muscle tissue of juvenile Atlantic salmon (Salmo salar) to compare their usefulness for reflecting specific growth rates, and to determine whether either growth index was influenced by diel variations or time of feeding. Three groups (n = 54 in total) were fed 1.5% of body weight in commercial pellets in four feedings per day. One group was fed only in the morning (0830–1230h), one in the afternoon (1430–1830h), and one in the morning and afternoon (0830–1830h). At the end of ten days, fish were sampled at three times (0130h, 1030h, 1630h) over a single 24h period. Correlations to specific growth rate were slightly higher for RNA concentrations than for ODC activity, but both were highly significant. RNA and ODC activity were also correlated to each other. These results suggest that RNA concentration and ODC activity, taken together, can be used to monitor changes in both the numbers and activity of ribosomes. For RNA concentrations, there was no evidence of an effect of diel variations or the time of feeding. For ODC activity, a significant diel effect (all feed schedules combined) was detected if one non-growing fish was excluded from the analysis; activity of the enzyme was slightly higher in the sample taken at night (0130h) than in the two daytime samples. Contribution no. 8, Catamaran Brook Habitat Research Project     

Dietary inclusion of crystalline D- and L-methionine: effects on growth, feed and protein utilization, and digestibility in small and large Atlantic salmon (Salmon salar L.)

Use of crystalline methionine to optimize amino acid profiles in feeds for Atlantic salmon (Salmo salar L.) was studied in three experiments. A basic experimental diet with 25% of dietary protein from marine origin and the remaining 75% from soya protein concentrate was used either with or without supplementation of L ‐methionine, D ‐methionine or DL ‐methionine. A fishmeal‐based diet was used as control. In experiment 1, growth performance of 100 g salmon in seawater was improved by supplementation with the different methionine products. Growth performance was, however, inferior to that of fish fed the control. Feed conversion ratios (FCR) exhibited similar trends to specific growth rate (SGR) with methionine supplementation, but again not as the control. Protein retention, measured as productive protein value (PPV), was significantly better in the control and D ‐methionine groups as compared with either L ‐methionine or the nonsupplemented group. These trends were confirmed in experiment 2, using fish of the same size, where an absorption and digestibility study of D ‐ and L ‐methionine clearly indicated that D ‐methionine was absorbed as well as L ‐methionine. Results indicated that racemization of D ‐ into L ‐methionine occurs in liver and kidney in Atlantic salmon. Ornithine decarboxylase (ODC) activity in liver was significantly affected by dietary treatment and there was a good correlation with growth and feed utilization data. In experiment 3, a growth study with large salmon (1 kg) was performed using increasing supplementation of DL ‐methionine to the basic diet. Results indicated a methionine requirement for maximum growth of 9.2 g kg^–1 feed dry matter (DM) kg^–1 growth. This is slightly higher than that reported in the literature. The present study clearly shows that optimizing the amino acid profile by inclusion of a single crystalline amino acid (crystalline methionine) does not give the same protein utilization as that of a diet in which the amino acids are mainly protein bound. D ‐methionine was found to result in better protein utilization than L ‐methionine when measured as PPV.     

α-乙酰乳酸脱羧酶基因在枯草芽孢杆菌中的整合型表达

α-乙酰乳酸脱羧酶在啤酒生产中能加快啤酒成熟,有重要的应用价值。本研究将枯草芽孢杆菌启动子P43克隆到质粒pUC19-ALDC中的α-乙酰乳酸脱羧酶基因之前,得到重组质粒pUC19-P43-ALDC。重组质粒pUC19-P43-ALDC与质粒pMLK83-BN同源重组,筛选得到枯草芽孢杆菌整合质粒pMLK83-ALDC。用此整合质粒转化枯草芽孢杆菌1A751,挑选出新霉素抗性且无淀粉酶活性的重组菌株。此菌株用LB培养基在37℃、220r/min摇瓶培养过夜,测得α-乙酰乳酸脱羧酶活力为15.6U/mL,说明整合的α-乙酰乳酸脱羧酶基因能够在重组菌株中稳定传代和表达。本研究首次在枯草芽孢杆菌中用整合型的方式重组表达了α-乙酰乳酸脱羧酶,提出了一种有潜力的生产α-乙酰乳酸脱羧酶的新方法。