脂肪酸及其氧合物对曲霉属真菌菌丝生长、产孢和黄曲霉毒素合成的影响

黄曲霉毒素是由黄曲霉菌合成的一类毒性极高、致癌性极强的次生代谢物。一般认为,高油脂含量的作物种子被曲霉属真菌感染后容易产生黄曲霉毒素,但是,脂肪酸的处理实验结果表明不同类型的脂肪酸对曲霉属真菌毒素合成的作用不同,有的促进合成,有的抑制合成。最近研究结果显示所有脂肪酸都促进黄曲霉毒素合成,但是多不饱和脂肪酸在暴露空气之后对毒素合成有抑制作用。这种抑制产毒的作用似乎是由多不饱和脂肪酸氧化所产生的脂氧合物所介导。本文结合我们的研究结果,综合评述了脂肪酸和脂氧合物调控曲霉属真菌菌丝生长、产孢和毒素合成研究的最新进展。     

Epoxyeicosatrienoic acids enhance axonal growth in primary sensory and cortical neuronal cell cultures

It has recently been reported that soluble epoxide hydrolase (sEH), the major enzyme that metabolizes epoxyeicosatrienoic acids (EETs), is expressed in axons of cortical neurons; however, the functional relevance of axonal sEH localization is unknown. Immunocytochemical analyses demonstrate predominant axonal localization of sEH in primary cultures of not only cortical but also sympathetic and sensory neurons. Morphometric analyses of cultured sensory neurons indicate that exposure to a regioisomeric mixture of EETs (0.01-1.0 μM) causes a concentration-dependent increase in axon outgrowth. This axon promoting activity is not a generalized property of all regioisomers of EETs as axonal growth is enhanced in sensory neurons exposed to 14,15-EET but not 8,9- or 11,12-EET. 14,15-EET also promotes axon outgrowth in cultured cortical neurons. Co-exposure to EETs and either of two structurally diverse pharmacological inhibitors of sEH potentiates the axon-enhancing activity of EETs in sensory and cortical neurons. Mass spectrometry indicates that sEH inhibition significantly increases EETs and significantly decreases dihydroxyeicosatrienoic acid metabolites in neuronal cell cultures. These data indicate that EETs enhance axon outgrowth and suggest that axonal sEH activity regulates EETs-induced axon outgrowth. These findings suggest a novel therapeutic use of sEH inhibitors in promoting nerve regeneration.     

Endocytotic elimination and domain-selective tethering constitute a potential mechanism of protein segregation at the axonal initial segment

The axonal initial segment is a unique subdomain of the neuron that maintains cellular polarization and contributes to electrogenesis. To obtain new insights into the mechanisms that determine protein segregation in this subdomain, we analyzed the trafficking of a reporter protein containing the cytoplasmic II-III linker sequence involved in sodium channel targeting and clustering. Here, we show that this reporter protein is preferentially inserted in the somatodendritic domain and is trapped at the axonal initial segment by tethering to the cytoskeleton, before its insertion in the axonal tips. The nontethered population in dendrites, soma, and the distal part of axons is subsequently eliminated by endocytosis. We provide evidence for the involvement of two independent determinants in the II-III linker of sodium channels. These findings indicate that endocytotic elimination and domain-selective tethering constitute a potential mechanism of protein segregation at the axonal initial segment of hippocampal neurons.     

Identification of biosynthetic precursors for the endocannabinoid anandamide in the rat brain

Anandamide is an endogenous signaling lipid that binds to and activates cannabinoid receptors in the brain and peripheral tissues. The endogenous precursors of anandamide, N-arachidonoyl phosphatidylethanolamines (NArPEs), are a family of complex glycerophospholipids that derive from the exchange reaction of an arachidonoyl group between the sn-1 position of phosphatidylcholine and the primary amine of phosphatidylethanolamine catalyzed by N-acyl transferase activity. A precise characterization of the molecular composition of NArPE species generating anandamide has not yet been reported. In the present study, using liquid chromatography coupled to electrospray ionization ion-trap mass spectrometry, we identified the major endogenous NArPE species, which mainly contained sn-1 alkenyl groups (C16:0, C18:0, C18:1) and monounsaturated (C18:1) or polyunsaturated (C20:4, C22:4, C22:6) acyl groups at the sn-2 position of the glycerol backbone. Using rat brain particulate fractions, we observed a calcium-dependent increase in both NArPEs and anandamide formation after incubation at 37 degrees C for 30 min. Furthermore, a targeted lipidomic analysis showed that Ca(2+) specifically stimulated the formation of PUFA-containing NArPE species. These results reveal a previously unrecognized preference of brain N-acyl transferase activity for polyunsaturated NArPE and provide new insights on the physiological regulation of anandamide biosynthesis.     

Changes occurring in cortical NO release and brain NO-synthases during a paradoxical sleep deprivation and subsequent recovery in the rat

Variations occurring in cortical nitric oxide (NO) release were analysed with a voltametric method in rats (i) placed in control conditions, (ii) while being paradoxical sleep deprived (PSD), or (iii) recovering from a PSD. Activities of neuronal (nNOS) and inducible (iNOS) NO-synthases as well as nNOS expression were also determined in several brain regions. In baseline conditions, circadian variations in nNOS expression and activity were maximal during the dark period and minimal during the light one for all the structures analysed (frontal cortex, pons and medulla). In the same way, cortical NO release occurred through a circadian rhythm exhibiting maxima and minima during dark and light periods, respectively. In the same experimental conditions, iNOS activity did not exhibit time-dependent changes. The correlative changes observed in baseline conditions between NO release, nNOS expression and activity within the frontal cortex were disrupted during PSD and subsequent recovery. Still again, iNOS activity remained unchanged. Results obtained point out that the tight coupling existing in control conditions between nNOS expression-activity and NO release is disrupted by a PSD and remains affected during the subsequent 24 h recovery. Their significance is discussed.     

Procedure for the sample preparation and handling for the determination of amino acids, monoamines and metabolites from microdissected brain regions of the rat

A method is described for the analysis of amino acids, monoamines and metabolites by high-performance liquid chromatography with electrochemical detection (HPLC–ED) from individual brain areas. The chromatographic separations were achieved using microbore columns. For amino acids we used a 100×1 mm I.D. C₈, 5 μm column. A binary mobile phases was used: mobile phase A consisted of 0.1 M sodium acetate buffer (pH 6.8)–methanol–dimethylacetamide (69:24:7, v/v) and mobile phase B consisted of sodium acetate buffer (pH 6.8)–methanol–dimethylacetamide (15:45:40, v/v). The flow-rate was maintained at 150 μl/min. For monoamines and metabolites we used a 150×1 mm I.D. C₁₈ 5 μm reversed-phase column. The mobile phase consisted of 25 mM monobasic sodium phosphate, 50 mM sodium citrate, 27 μM disodium EDTA, 10 mM diethylamine, 2.2 mM octane sulfonic acid and 10 mM sodium chloride with 3% methanol and 2.2% dimethylacetamide. The potential was +700 mV versus Ag/AgCl reference electrode for both the amino acids and the biogenic amines and metabolites. Ten rat brain regions, including various cortical areas, the cerebellum, hippocampus, substantia nigra, red nucleus and locus coeruleus were microdissected or micropunched from frozen 300-μm tissue slices. Tissue samples were homogenized in 50 or 100 μl of 0.05 M perchloric acid. The precise handling and processing of the tissue samples and tissue homogenates are described in detail, since care must be exercised in processing such small volumes while preventing sample degradation. An aliquot of the sample was derivatized to form the tert.-butylthiol derivatives of the amino acids and γ-aminobutyric acid. A second aliquot of the same sample was used for monamine and metabolite analyses. The results indicate that the procedure is ideal for processing and analyzing small tissue samples.     

Fibroblast growth factor 2 induces apoptosis in the early primary culture of rat cortical neurons

In the central nervous system, fibroblast growth factor 2 (FGF2) is known to have important functions in cell survival and differentiation. In addition to its roles as a neurotrophic factor, we found that FGF2 caused cell death in the early primary culture of cortical neurons. FGF2-induced neuronal cell death showed apoptotic characters, e.g., chromatin condensation and DNA fragmentation. The ultrastructural morphology of FGF2-treated neurons indicated apoptotic features such as progressive cell shrinkage, blebbing of the plasma membrane, loss of cytosolic organelles, clumping of chromatin, and fragmentation of DNA. Tyrosine kinase inhibitors significantly rescued neurons from FGF2-induced apoptosis. FGF2 potentiated a marked influx of Ca²⁺ into neurons before apoptosis. Both a calcium chelator and L-type voltage-sensitive Ca²⁺ channel (L-VSCC) blockers attenuated FGF2-induced apoptosis, whereas other blockers of VSCCs such as N-type and P/Q-types did not. Blockers of L-VSCCs significantly suppressed FGF2-enhanced Ca²⁺ influx into neurons. Moreover, FGF2 also generated reactive oxygen species (ROS) before apoptosis. Radical scavengers reduced not only the FGF2-generated ROS, but also the FGF2-induced Ca²⁺ influx and apoptosis. In conclusion, we demonstrated that FGF2 caused apoptosis via L-VSCCs in the early neuronal culture.     

A method for the direct evaluation of the fatty acid status in a drop of blood from a fingertip in humans: applicability to nutritional and epidemiological studies

Several studies have shown that the fatty acid composition of circulating lipids reflects dietary fat intake, in turn being related to health status. The fatty acid composition of plasma lipids is therefore an important parameter in studies on dietary interventions. The aim of our study was to develop a rapid and inexpensive method for the analysis of circulating fatty acids applicable to large population groups. Drops of blood collected from fingertips have been directly subjected to transmethylation for gas chromatography analysis. This new method, validated for reproducibility, has been compared with the conventional method, based on withdrawal of blood from the antecubital vein followed by lipid extraction, and identical data have been obtained with the two techniques. Observed and predicted differences between blood and plasma fatty acids are related to the contribution of circulating cell membranes in blood. Finally the application of the methods to samples from 100 healthy subjects and the assessed correlation between dietary habits and blood fatty acid profiles demonstrate the validity of the new method and its applicability to nutritional and epidemiological studies.     

小鼠肠道菌群代谢产物与糖尿病的相关性研究

目的研究糖尿病小鼠粪便中肠道菌群代谢产物与血糖之间的相关性,探讨肠道菌群与糖尿病之间的关系。方法采用高脂饮料喂养加腹腔注射链脲佐菌素(STZ)的方法建立糖尿病小鼠模型;将实验动物随机分为正常组、高脂组、糖尿病组及模型给药组,连续给药5周后,采血测血糖血脂,同步收集动物粪便,测粪便中短链脂肪酸(Short-chain fatty acids,SCFA)及D-乳酸。SCFA的检测使用气相色谱法,D-乳酸的检测使用紫外酶促法。结果糖尿病组小鼠粪便中乙酸、丙酸和正丁酸含量明显低于正常组及高脂组(P<0.01),D-乳酸含量明显高于正常组及高脂组(P<0.01);给药组乙酸、丙酸和正丁酸含量明显高于糖尿病组(P<0.01),D-乳酸含量明显低于糖尿病组(P<0.01)。给药组丙酸、正丁酸的含量与正常组间差异无统计学意义(P>0.05),但乙酸的含量仍低于正常组(P<0.01),D-乳酸的含量仍高于正常组(P<0.01)。结论糖尿病小鼠粪便中的肠道菌群代谢产物与血糖之间存在着密切的关系,代谢产物的差异性,提示肠道菌群的差异性,反映出糖尿病小鼠存在肠道菌群紊乱。     

Neural 22-Carbon Fatty Acids in the Weanling Rat Respond Rapidly and Specifically to a Range of Dietary Linoleic to of-Linolenic Fatty Acid Ratios

Changing the dietary ratio of the essential fatty acids (EFA), 18:2n6 and 18:3n3, while keeping the amounts of other fatty acids in the diet constant can rapidly and specifically alter the proportions of n6 and n3 22-carbon fatty acids in the brain of the weanling rat. A dietary 18:2n6/18:3n3 ratio of 165 versus 1.8 caused higher n6 and lower n3 22-carbon fatty acid levels, without changing total 22-carbon fatty acid levels, in phosphatidylethanolamine and phosphatidylcholine from several neural membrane fractions. This was apparent after only 2 weeks and showed no sign of plateauing after 12 weeks. Other neural fatty acids were essentially unaffected. The three most abundant 22-carbon fatty acids responded somewhat differently to increments in the dietary 18:2n6/18:3n3 ratio (1.8, 9, 36, and 165). Levels of 22:4n6 increased by similar absolute amounts for each four-fold increase in dietary 18:2n6/18:3n3 ratio; in contrast, the largest absolute changes in 22:5n6 and 22:6n3 levels occurred as the 18:2n6/18:3n3 ratio increased from 36 to 165. This study shows that the 18:2n6/18:3n3 ratio of diets high in fat (40% of energy) and adequate in EFA, both typical of diets in developed countries, can substantially and relatively quickly affect the 22-carbon fatty acids in the brain, even after the rapid accumulation of these fatty acids during neural growth has ceased.     

Differences in dietary and plasma fatty acids between wild and captive populations of a rare reptile, the tuatara (Sphenodon punctatus)

Tuatara (Sphenodon) are rare reptiles endemic to New Zealand. Wild tuatara on Stephens Island (study population) prey on insects as well as the eggs and chicks of a small nesting seabird, the fairy prion (Pachyptila turtur). Tuatara in captivity (zoos) are fed diets containing different insects and lacking seabirds. We compared the fatty acid composition of major dietary items and plasma of wild and captive tuatara. Fairy prions (eaten by tuatara in the wild) were rich in C20 and C22 polyunsaturated fatty acids (PUFA), especially the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In contrast, items from the diet of captive tuatara contained no C20 and C22 PUFA and were higher in medium-chain and less unsaturated fatty acids. Plasma from wild tuatara was higher in n-3 PUFA [including alpha-linoleic acid (C18:3n-3), EPA and DHA], and generally lower in oleic acid (C18:1) and palmitic acid (C16:0), than plasma from captive tuatara in the various fractions (phospholipid, triacylglycerol, cholesterol ester and free fatty acids). Plasma from wild adult tuatara showed strong seasonal variation in fatty acid composition, reflecting seasonal consumption of fairy prions. Differences in the composition of diets and plasma between wild and captive tuatara may have consequences for growth and reproduction in captivity. Accepted: 3 August 1998     

Prostaglandin synthesis in rat brain astrocytes is under the control of the n-3 docosahexaenoic acid, released by group VIB calcium-independent phospholipase A2

In the current study, we reveal that in astrocytes the VIB Ca(2+)-independent phospholipase A(2) is the enzyme responsible for the release of docosahexaenoic acid (22:6n-3). After pharmacological inhibition and siRNA silencing of VIB Ca(2+)-independent phospholipase A(2), docosahexaenoic acid release was strongly suppressed in astrocytes, which were acutely stimulated (30 min) with ATP and glutamate or after prolonged (6 h) stimulation with the endotoxin lipopolysaccharide. Docosahexaenoic acid release proceeds simultaneously with arachidonic acid (20:4n-6) release and prostaglandin liberation from astrocytes. We found that prostaglandin production is negatively controlled by endogenous docosahexaenoic acid, since pharmacological inhibition and siRNA silencing of VIB Ca(2+)-independent phospholipase A(2) significantly amplified the prostaglandin release by astrocytes stimulated with ATP, glutamate, and lipopolysaccharide. Addition of exogenous docosahexaenoic acid inhibited prostaglandin synthesis, which suggests that the negative control of prostaglandin synthesis observed here is likely due to competitive inhibition of cyclooxygenase-1/2 by free docosahexaenoic acid. Additionally, treatment of astrocytes with docosahexaenoic acid leads to the reduction in cyclooxygenase-1 expression, which also contributes to reduced prostaglandin production observed in lipopolysaccharide-stimulated cells. Thus, we identify a regulatory mechanism important for the brain, in which docosahexaenoic acid released from astrocytes by VIB Ca(2+)-independent phospholipase A(2) negatively controls prostaglandin production.     

Effects of hepatocyte growth factor (HGF) and activin a on the morphogenesis of rat submandibular gland-derived epithelial cells in serum-free collagen gel culture

Summary To study the mechanisms of morphogenesis in salivary gland regeneration, we have established the RSMG-1 cell line derived from submandibular gland (SMG) of 10-wk-old Wistar female rats in serum-free culture. Our finding that RSMG-1 cells originated from duct cells was based on morphology and immunohistochemical results. In three-dimensional serum-free collagen gel culture, HGF induced branching morphogenesis of RSMG-1 cells. Histological examination revealed that HGF-induced branching structure exhibited well-formed lumina. This morphology closely resembles that found in vivo. The cells also expressed activin A. Exogenously added activin A at a high concentration reduced HGF-induced branching morphogenesis. These findings suggest that the morphogenesis of the salivary gland is modulated by HGF and activin A. Our results show that the RSMG-1 cell line may be useful in studies of salivary gland regeneration.     

Effect of aging on monoamines and their metabolites in the rat brain

Concentrations of dopamine (DA), norepinephrine (NE), serotonin (5-HT) and their acid merabolites were assayed in specific brain areas of Wistar rats of various ages. DA and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were significantly lower in striatum and mesolimbic areas of old (24 mos) rats than young adult (3 mos), but not mature (12 mos) rats. The decrease of homovanillic acid (HVA) was significant in mesolimbic areas but not in striatum. Neither cortical NE nor its metabolite methoxydroxyphenylglycol sulphate (MHPG-SO₄) were significantly changed by aging. 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in the brainstem showed a tendency to a decrease and increase respectively in aged animals compared with young adults, but the differences were not statistically significant. However, the ratio of 5-HIAA to 5-HT concentrations was significantly higher in aged animals. The conclusion can be drawn that, in these brain areas, DA is more vulnerable to aging than NE and 5-HT, the metabolism of the latter being even enhanced.     

Effects of polyunsaturated fatty acids and gonadotropin on prostaglandin series E production in a primary testis cell culture system for the European sea bass

Prostaglandin series E (PGE) production by mixed populations of isolated sea bass Dicentrarchus labrax testicular cells was measured after culture at 20° C in L-15 for up to 24 h in the absence or presence of arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and/or gonadotropin (hCG). AA stimulated a significant dose- and time-dependent increase in PGE production. EPA stimulated PGE production only during the first 6 h although levels were 26·4% those induced with AA; DHA reduced PGE production. Although hCG alone had no effect, it enhanced AA-induced PGE formation and suppressed EPA-induced PGE production. These results suggest that essential PUFAs modulate significantly testicular PGE production in vitro and therefore may have important effects on steroidogenesis and spermiation in the male European sea bass.     

Pharmacokinetics of systemically administered tyrosine: a comparison of serum, brain tissue and in vivo microdialysate levels in the rat

Tyrosine uptake has been reported to differ across brain regions. However, such studies have typically been conducted over brief intervals and in anesthetized rats; anesthesia itself affects amino acid transport across the blood-brain barrier. To address these concerns, serum, brain tissue and in vivo microdialysate tyrosine levels were compared for 0-3 h after administration of tyrosine [0.138-1.10 mmol/kg intraperitoneally (i.p.)] to groups of awake rats. Serum and brain tissue tyrosine levels increased linearly with respect to dose. Basal tissue tyrosine levels varied significantly across brain regions [medial prefrontal cortex (MPFC), striatum, hypothalamus, and cerebellum], but the rate of tyrosine uptake was similar for hypothalamus, striatum and MPFC. For brain regions in which tyrosine levels in both microdialysate and tissue were assayed, namely MPFC and striatum, there was a high degree of correlation between tyrosine levels in tissue and in microdialysate. Increasing brain tyrosine levels had no effect on DA levels in MPFC microdialysate. We conclude that (i) regional differences in the response of dopamine neurons to systemic tyrosine administration cannot be attributed to pharmacokinetic factors; (ii) in vivo microdialysate provides an excellent index over time and across a wide range of tyrosine doses, of brain tissue tyrosine levels; and (iii) increases in brain tyrosine levels do not affect basal DA release in the MPFC.     

Triiodothyronine accelerates the synthesis of Synapsin I in developing neurons from fetal rat brain cultured in a synthetic medium

The effect of Triiodothyronine (T3) on Synapsin I appearance in rat cortical neurons has been investigated in vitro. Neuronal cultures from 16-day-old fetal rat brain grown in the absence of T3, express immunohystochemically detectable Synapsin I at the 14th day in vitro. The addition of the hormone to the culture medium determines an early (at the 7th day in vitro) appearance of fluorescent dots specific for Synapsin I.