鞘糖脂研究进展

鞘糖脂位于细胞膜脂质双分子层,是哺乳动物细胞膜上的必需组成成分之一,参与细胞的多种生物学活动,其生物学功能非常复杂。在免疫应答、细胞发育、细胞识别及分化中都发挥重要的作用。组织器官中鞘糖脂的异常表达往往与各种疾病具有明显的相关性。因此鞘糖脂的结构表征、构效关系研究以及与之相关的鞘糖脂生物合成及分解代谢途径的研究已成为近年来的热点问题。最近研究也表明鞘糖脂在疾病的发生发展及治疗过程中都具有至关重要的作用。对鞘糖脂的结构与功能的关系、糖脂的合成代谢以及糖脂的分离分析方法作一综述。     

人子宫平滑肌肉瘤中性鞘糖脂含量、组成及其四糖基组分糖链结构的变化

本文分析了人子宫平滑肌肉瘤组织的中性鞘糖脂,发现子宫肉瘤组织的中性鞘糖脂的含量明显低于正常组织,组成成分虽与正常子宫平滑肌相似,均含有单、双、三、四糖基及多糖基组分,但各组分的相对含量则变化显著。肉瘤中所含短糖链的组分相对减少,高极性的CPH明显增加。本文还纯化了子宫肉瘤中性糖脂中主要的四糖基组分,应用HPTLC、酸解和酶解法以及特异单抗放射免疫染色法对该组分进行鉴定。结果证明子宫平滑肌中所含主要的四糖基组分糖链结构在正常组织为Globo系列的红细胞苷脂,而在肉瘤中则转变为乳糖系列的拟红细胞糖苷脂。     

鞘糖脂的新陈代谢与生理学功能（英文）

鞘糖脂由一个神经酰胺的脂骨架与一个或多个糖基连接形成,存在于细胞膜中,承担多种生理学功能。我们将对鞘糖脂的生物化学及生理学方面研究做一概述,随后简要介绍近年来鞘糖脂的临床研究进展。在讨论鞘糖脂生物化学方面的研究中,我们把重点放在介绍鞘脂类及鞘糖脂的结构和生合成途径。脂类生物合成和降解是通过一系列酶的参与紧密调控的,如果一种酶参与代谢失败会导致酶底物的大量堆积,会引起溶酶体贮积症,这种疾病是由具有分解代谢活性的水解酶缺失所造成的。随后,我们介绍鞘糖脂在细胞及动物体内的生理学方面的功能,以及鞘糖脂在临床方面的一些病症中所起的作用,即使许多细节还有待于进一步研究。     

人体子宫内膜中性鞘糖脂和神经节苷脂的含量和组成在月经周期中和妊娠时的变化

从月经周期各期、早妊(6—8周)及足月妊娠子宫内膜中提取、纯化了神经节苷脂(Gls)和中性鞘糖脂(N-GSL),分别测定了其含量。对两类鞘糖脂的组成进行了HPTLC图谱分析。初步观察了不同时期子宫内膜中CMP—NeuAc:LacCer唾液酰转移酶(ST_1)和CMP-NeuAc:CM_3唾液酰转移酶(ST_2)的活性。结果表明:分泌期的GLs总含量低于增生期(P<0.02);分泌期GD_3含量较生长期增多(P<0.01);妊娠后,GM_3含量增加,而GD_3减少;相应地,ST_1活性增高,ST_2活性降低。分泌期CMH含量为增生期的4.7倍。结果提示子宫内膜鞘糖脂含量和组成的变化可能与子宫的功能有关,而且受女性激素水平的影响。     

妊娠期家兔子宫内膜的鞘糖脂表达

 妊娠期家兎子宫内膜的神经节苷脂(Gls)的含量明显低于动情期的,而中性鞘糖脂(NGSL)的含量则以妊娠中、晚期的最高,动情期最低。鞘糖脂组成变化最显著的是妊娠早期,由动情期到早孕GM_3从28.0％增加到52.7％,CMH.CDH由未测出分别增加到29.2％和21.9％,而糖链复杂的组分GD_3,GTlb和CPH的百分含量则明显减少,到妊娠中、晚期、短糖链组分逐渐减少,而复杂糖链组分渐增。中期妊娠内膜的(GIs)以GD_3为主要组分,占45％,明显高于其它各期。NGSL在妊娠中、晚期CPH增高达70％,与动情期水平相当。结果提示,妊娠期间子宫内膜的鞘糖脂含量与组成均发生明显变化,这些变化可能与子宫功能密切相关。特别是早孕对的变化,推测与子宫内膜和胚泡的识别,粘连特性的获得有关。     

GM3对J6-2细胞的促分化作用及对鞘糖脂与磷脂组分的影响

本文在用形态学与组化手段证实神经节苷脂GM3促进国人白血病细胞系J6-2分化的基础上,研究了分化前后细胞鞘糖脂及磷脂组分的变化。作者用自行提纯的GM3表明,J6-2细胞在分化前后没有显著的神经节苷脂组分的改变,中性鞘糖脂组分CMH在分化后明显增多。GM3使LPC、SPM及PI+PS等磷脂组分减少,但使PC组分增多。此项结果提示了神经节苷脂的一个新的功能——调节磷脂代谢,但其具体机制尚待阐明。     

糖苷合酶应用于糖蛋白和鞘糖脂的合成

糖苷合成酶是糖苷酶的亲核体氨基酸突变酶,催化寡糖的高效合成,可应用于寡糖的大规模生产。最近,糖苷合成酶被成功地应用于两类重要的生物分子——糖蛋白和鞘糖脂的高效合成,这必将对糖生物学和制药业的发展起到重要的推动作用。     

狗小肠鞘氨醇糖脂中长链碱组成的研究

 利用气相色谱、气相色谱-质谱联用等方法测定了狗小肠鞘氨醇糖脂中的长链碱组成。其主要的长链碱为鞘氨醇(Sphingosine)、异鞘氨醇(isosphingosine)、二氢鞘氨醇(Sphinganine)和植物鞘氨酸(Phyto-sphingosine)。一共分离出十三个鞘氨醇糖脂。在唯一的五糖基神经酰胺中异鞘氨醇是主要成份。在一个一糖基神经酰胺中植物鞘氨醇是主要成份。植植物鞘氨酸也是两个二糖基神经酰胺和一个三糖基神酰胺的主要长链碱。说明它不仅存于植物体内。     

人正常子宫肌肉的鞘糖脂组成及其在不同生理功能阶段的变化

本文测定了新生儿、生育期、更年期和足月妊娠人子宫肌肉的神经节苷脂(Gg)与中性鞘糖脂(N-GSL)的含量,比较了两种鞘糖脂的HPTLC谱。新生儿期Gg的总含量(以脂结合唾液酸LBSA量表示)最高,每克湿重组织约45.2μg,足月妊娠子宫肌肉中的含量最低,为10.4μg,生育期为32.8μg、更年期为39.5μg。N—GSL的含量却以足月妊娠子宫肌肉中最多,达99.4μg。按HPTLC谱分析子宫肌肉中Gg的主要组分为GD_3和GM_3,在子宫发育成熟与妊娠时,肌肉组织中这两种组分的含量变化明显:生育期样品的GD_3由新生儿的25.4％增加到56.6％(按占LBSA总量的百分比计算),GM_3则由33.2％降至16.9％。此外,GM_1和GD_(1a)也明显减少。N—GSL在生育期CMH、CDH和CTH的含量(按占含糖基量的百分比计算)成倍增加,而含五糖基以上的组分则仅为新生儿子宫的1/5。足月妊娠与新生儿子宫肌肉的两类鞘糖脂的HPTLC谱类似,但前者GT1b占19.4％,明显高于新生儿样品(6.1％)。     

雌、孕激素对去卵巢家兔子宫内膜鞘糖脂表达的影响

用雌、孕激素处理去卵巢家兔后观察子宫内膜鞘糖脂的含量和组成。结果表明、雌激素处理后、子宫内膜Gls含量是孕激素处理的十余倍,而孕激素增加NGSL含量的作用却比雌激素更强。两种激素对鞘糖脂组分的影响也明显不同。雌激素给药组子宫内膜鞘糖脂以多糖基组分GD_3 GT_(1b) CPH为主,孕激素组则以短糖链组分GM_3 CMH CDH为主。雌激素预先作用后再用孕激素或雌、孕激素同时给药,与单用雌激素后比较其短糖链组分明显增加,多糖基组分则明显减少,表现出两者的共同作用。上述结果提示:雌、孕激素对鞘糖脂代谢影响明显不同,雌激素使糖链复杂化、孕激素则使糖链趋向于简单。     

Cationic Glycosphingolipids in Neuronal Tissues and Their Possible Biological Significance

During the course of studies on natural occurrence of sphingosine base in brain, cationic glycosphingolipids bound to carboxymethyl-Sephadex and eluted with triethylamine in organic solvents were isolated and characterized. Four classes of compounds were identified: (i) plasmalopsychosine-A and -B; (ii) glyceroplasmalopsychosine; (iii) glycosphingolipids having de-N-acetyl-hexosamine, e.g., de-N-acetyl-Lc₃Cer; (iv) glycosylsphingosine, i.e., lysoglycosphingolipid. Only two kinds, galactosylsphingosine (psychosine) and lactosylsphingosine, were found to occur naturally in brain. All these compounds were isolated from extract of brain white matter. Their occurrence, quantity, and distribution pattern differ from one species to another. Their quantity is much lower than that of regular acidic and neutral glycosphingolipids. They may interact with regular glycosphingolipids in glycosphingolipid-enriched microdomains to elicit signal transduction, to modify cellular phenotype, although studies along this line are highly limited at this time.     

Gene cloning and enzyme structure modeling of the <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> N74 fructosyltransferase

The fructooligosaccharides (FOS) represent an important source of prebiotic compounds that are widely used as an ingredient in functional foods. Recently, the strain Aspergillus oryzae N74 was reported as a potential microorganism for the industrial production of FOS, due to its high yields of FOS production. In this work, we used a PCR-cloning strategy to clone the A. oryzae N74 ftase gene as a previous step for recombinant enzyme production. Ftase showed a 1630 bp size with a 99% similarity with other A. oryzae strains and between 1 to 68% identities with other Aspergillus strains. This gene encodes for a 525 amino acids protein with 99% similarity with other A. oryzae strains and between 11 to 69% similarities with other Aspergillus strains. Finally, an A. oryzae N74 FTase tertiary structure model was predicted base on its similarity with other glycoside hydrolase 32 family members. The active site was located inside the β-propeller domain and was formed for non-charged polar and charged amino acids. In summary, these results shows the high level of sequence conservation between A. oryzae strains and represent a first step towards the development of a FOS production industrial process using recombinant microorganism carrying the ftase gene from A. oryzae N74.     

Interaction of myelin basic protein, melittin and bovine serum albumin with gangliosides, sulphatide and neutral glycosphingolipids in mixed monolayers

Some parameters that may regulate the miscibility and stability of mixed lipid-protein monolayers at the air-145 mM NaCl interface were studied employing six glycosphingolipids (acidic or neutral), three different types of proteins (soluble, extrinsic or highly amphipathic) and some phospholipids. The results obtained show that the percentage of the total area occupied by the protein at the interface is an important parameter leading to lateral phase separations; the amount and area contribution of the protein accepted in the film before the components become immiscible increase with the complexity of the polar head group of the glycosphingolipids. The interactions occur with progressive reductions of the intermolecular packing as the polar head group of the glycosphingolipid becomes more complex and this is accompanied by more negative values of the excess free energy of mixing. The lipid component seems to be the major responsible for the reduction in mean molecular area.     

Metabolism of the Soy Isoflavones Daidzein and Genistein by Fungi Used in the Preparation of Various Fermented Soybean Foods

The ability of fungi used in the preparation of fermented soybean foods to metabolize the soy isoflavones daidzein and genistein was investigated. A total of 21 fungal strains from dou-chi, miso, sake, soy sauce, and sufu were screened. The genera of the tested fungi included Actinomucor, Aspergillus, Candida, Debaryomyces, Monascus, Mucor, Rhizopus, Saccharomyces, and Zygosaccharomyces. The results were that all tested Aspergillus strains from these soybean foods, including five A. oryzae strains, one A. sojae strain, and one A. tamarii strain, metabolized both daidzein and genistein. In contrast, no other tested fungi from the fermented soybean foods metabolized either daidzein or genistein. The metabolites of daidzein and genistein by Aspergillus strains were identified as 8-hydroxydaidzein and 8-hydroxygenistein, respectively, based on their mass, ¹H-, and ¹³C-NMR spectra.     

米曲霉异源蛋白表达系统研究进展及展望

米曲霉作为一种重要的工业微生物,在异源蛋白表达方面已有广泛应用,受限于被表达蛋白的修饰及分泌过程,目前实际生产使用的基因供体主要局限于其他真菌,尤其是丝状真菌。当外源基因来源于植物、昆虫和哺乳动物时,米曲霉所生产的异源蛋白产量及生物活性往往不尽如人意。本文综述了米曲霉作为宿主表达异源蛋白的研究进展,包括其现有的遗传操作手段及异源表达方面的应用及探索,重点介绍了应用过程中面临的挑战和解决策略,另外,对米曲霉表达异源蛋白的应用前景及发展方向进行了展望。     

A rapid preparative method for isolation of neutral and acidic glycosphingolipids by radial thin-layer chromatography

An efficient method to separate neutral and acidic glycosphingolipids (GSLs) from their mixtures within a short period (45-60 min) and with low consumption of solvents (chloroform-methanol-water, 60/35/8 (v/v/v); 250-500 ml) has been developed. This method utilizes a centrifugal thin-layer chromatograph (Chromatotron) and the GSL mixtures (30-400 mg) are applied to glass plates coated with a 1-mm layer of silica gel 60 PF-254. The method (radial thin-layer chromatography) is rapid and simple and the recovery of glycosphingolipids is high (70-80%).     

Impact of Aspergillus oryzae genomics on industrial production of metabolites

Aspergillus oryzae is used extensively for the production of the traditional Japanese fermented foods sake (rice wine), shoyu (soy sauce), and miso (soybean paste). In recent years, recombinant DNA technology has been used to enhance industrial enzyme production by A. oryzae. Recently completed genomic studies using expressed sequence tag (EST) analyses and whole-genome sequencing are quickly expanding the industrial potential of the fungus in biotechnology. Genes that have been newly discovered through genome research can be used for the production of novel valuable enzymes and chemicals, and are important for designing new industrial processes. This article describes recent progress of A . oryzae genomics and its impact on industrial production of enzymes, metabolites, and bioprocesses.     

B-group vitamins production by mycorrhizal fungi in response to pH (in vitro studies)

Studies were carried out on B-group vitamins production by mycorrhizal fungi grown in vitro at different pH values. It was found that not all the fungi investigated produced all the B-group vitamins studied. Production of the vitamins varied between species and was influenced by the pH of the medium. Out of seven fungal species studied three did not produce biotin. Suillus bovinus synthesized this vitamin both in the acidic and neutral medium. Thiamin was produced by the fungi in minute amounts mainly in the acidic medium. The greatest amounts of nicotinic acid were produced by Hebeloma crustuliniforme (No 5392). Pantothenic acid was not detected only in the culture of Cenococcum graniforme.     

Sphingolipid composition of human platelets

Total lipid extracts from washed trypsinized human platelets were fractionated into neutral lipids, glycosphingolipids, and phospholipids by silicic acid chromatography. The concentrations and chemical structures of the neutral and acidic glycosphingolipids were then studied in detail. On the basis of sugar molar ratios, studies of permethylation products, and the action of stereospecific glycosidases on the lipids, identifications were made of four neutral glycosphingolipids. Lactosylceramide was the most abundant type and accounted for 64% of the total neutral glycolipid mixture. The major fatty acids of the lactosylceramide were 20:0, 22:0, 24:0, and 24:1; the major long-chain base was 4-sphingenine. The platelets were surprisingly rich in a ceramide fraction, which represented 1.3% of the total platelet lipids. It had a different fatty acid composition than the neutral glycosphingolipid and ganglioside fractions. Hematoside was also isolated from the total lipid fraction of platelets; the neuraminic acid component was N-acetylneuraminic acid. Treatment of platelets with trypsin, chymotrypsin, or thrombin increased the yield of hematoside as compared with a control, while the level of ceramides was not changed. It was concluded that the platelets are similar to leukocytes, liver, and spleen in that lactosylceramide and hematoside are the principal neutral and acidic glycosphingolipids. The presence of a relatively high proportion of ceramide in platelets may be a unique characteristic of this cellular fraction of blood.     

Large‐scale molecular genetic analysis in plant‐pathogenic fungi: a decade of genome‐wide functional analysis

Plant‐pathogenic fungi cause diseases to all major crop plants world‐wide and threaten global food security. Underpinning fungal diseases are virulence genes facilitating plant host colonization that often marks pathogenesis and crop failures, as well as an increase in staple food prices. Fungal molecular genetics is therefore the cornerstone to the sustainable prevention of disease outbreaks. Pathogenicity studies using mutant collections provide immense function‐based information regarding virulence genes of economically relevant fungi. These collections are rich in potential targets for existing and new biological control agents. They contribute to host resistance breeding against fungal pathogens and are instrumental in searching for novel resistance genes through the identification of fungal effectors. Therefore, functional analyses of mutant collections propel gene discovery and characterization, and may be incorporated into disease management strategies. In the light of these attributes, mutant collections enhance the development of practical solutions to confront modern agricultural constraints. Here, a critical review of mutant collections constructed by various laboratories during the past decade is provided. We used Magnaporthe oryzae and Fusarium graminearum studies to show how mutant screens contribute to bridge existing knowledge gaps in pathogenicity and fungal–host interactions.