滇重楼寄生菌的研究

从滇重楼(Paris polyphylla var.yunnanensis)地下茎中分离和鉴定出两种细菌——蜡状芽孢杆菌(Bacillus cereus)和产碱假单胞菌(Pseudomonas alcaligenes),以及三种真菌——黑团孢霉(Periconia sp.)、白色厚顶孢霉(Pachnocybe albida)和重楼索霉(Hormomyces paridiphilus)。对蜡状芽孢杆菌、产碱假单胞菌和重楼索霉进行了液体培养并测定了胞外多糖含量,结果表明重楼索霉可分泌大量胞外多糖,这可能是导致滇重楼地下茎胶质化和多糖含量增加的原因。     

嗜盐隐杆藻胞外多糖的分离、纯化及理化特性

嗜盐隐杆藻（Ａｐｈａｎｏｔｈｅｃｅ ｈａｔｏｐｈｙｔｉｃａ）培养液经离心,浓缩、透析、有机溶剂沉淀得胞外多糖（Ｅｘｏｐｏｌｙｓａｃｃｈａｒｉｄｅｓ,ＥＰＳ）粗品,经ＤＥＡＥ－纤维素二次柱层析纯化得ＥＰＳ精品。葡聚糖Ｇ－２００凝胶过滤表明其为单一组份。对其进行理化测试并对各组分进行定量分析,多糖、已糖醛酸、硫酸根含量分别为４０．９６％２３．２７％和３４．４６％,元素分析你测得Ｃ、Ｈ、Ｎ、Ｓ含量分别为     

蛹虫草菌产生的胞外多糖及其理化性能和抗氧化活性的初步研究

蛹虫草菌Cordycepsmilitaris在液体发酵条件下可产生胞外多糖,其粗多糖的蛋白含量在14．31％左右,氨基酸含量为13．8％,共有17种氨基酸,水分5％左右,多糖含量大约占80％。蛹虫草菌胞外多糖具有良好的增稠性、触变性、抗盐耐热和对广泛pH的稳定性能。蠕虫草菌丝体及其胞外多糖具有一定的抗氧化活性,在60℃的促进氧化条件下放置7天,0．05％含量的菌丝体及其胞外多糖降低油脂氧化率分别达到18．8％和19．8％。     

红景天根中总黄酮和多糖的微波提取及含量分析

目的;从红景天中提取有效成分总黄酮,多糖,并测定其含量。方法：分光光度法,结果：测得红景天总黄酮含量4．62％,平均回收率为98．59％,RSD1．23（n=3),多糖含量9．12％,平均回收率为99．25％,RSD1．12％（n=3)。结论：运用微波技术辅助测定红景天中总黄酮和多糖的含量,反应速度加快,收率提高。     

浒苔多糖的分离、纯化和分析

浒苔（Ｅｎｔｅｒｏｍｏｒｐｈａｐｒｏｌｉｆｅｒａ）经热水提取,Ｓｅｖａｇｅ法除去蛋白质,用乙醇沉淀,ＳｅｐｈａｄｅｘＧ－１００柱层析,得浒苔多糖（简称ＥＰ）精制品。经ＳｅｐｈａｄｅｘＧ－２００柱层析鉴定为单一对称性洗脱峰。红外光谱分析具有多糖特征吸收峰,紫外光谱分析未见有核酸和蛋白质的特征吸收峰。总糖含量为８８．８％,其中糖醛酸含量为３３．６％。单糖组成为Ｌ－阿拉伯糖、Ｌ－岩藻糖、Ｄ－甘露糖、Ｄ－半乳糖及Ｄ－葡萄糖,平均分子量为２５０００。     

枸杞子糖蛋白的分离纯化,物化性质及糖肽键特征

从宁夏枸杞子提取得到的粗多糖,经ＤＥＡＥ－Ｃｅｌｌｕｌｏｓｅ和ＳｅｐｈａｄｅｘＧ－１００柱层析,得到均一的枸杞子糖蛋白ＬｂＧＰ。分子量由ＳＤＳ－ＰＡＧＥ测定为８８ｋｄ,糖含量为７０％,糖组成为Ａｒａ：Ｇａｌ：Ｇｌｃ＝２．５：１．０：１．０,并含有其他１８种天然氨基酸。初步分析表明ＬｂＧＰ是Ｏ－连接的糖蛋白。     

浒苔多糖的分离,纯化和分析

浒苔经热水提取,Ｓｅｖａｇｅ法除去蛋白质,用乙醇沉淀,Ｓｅｐｈａｄｅｘ－Ｇ－１００柱层析,得浒苔多糖（简称ＥＰ）精制品。经ＳｅｐｈａｄｅｘＧ－２００柱导析鉴定为单一对称性洗脱峰。红外光谱分析具有多糖特征吸收峰,紫外光谱分析表未见有核酸和蛋白质的特征及吸上峰,总糖含量为８８．８％,基中糖酸酸含量为３３．６％。单糖组成为Ｌ－阿拉伯糖,Ｌ－岩藻糖,Ｄ－甘露糖,Ｄ－半乳糖及Ｄ－葡萄糖。平均分子量为２５００     

深层培养云芝菌丝体蛋白多糖的提取及性质

采用水、３％草酸和０．２ｍｏｌ／Ｌ氢氧化钠为溶剂分别提取深层培养的云芝〔Ｐｏｌｙｓｔｉｃｔｕｓｖｅｒｓｉｃｏｌｏｒ（Ｌ．）Ｆｒ．〕菌丝体蛋白多糖,其得率分别为菌丝体干重的２０％、４９％和１９％。提取的粗多糖经ＳｅｐｈａｄｅｘＧ１００柱层析进一步分离、纯化,纯化的多糖经聚丙烯酰胺凝胶电泳分离后呈现一个斑点,在ＳｅｐｈａｄｅｘＧ１００柱层析中有一个对称峰,紫外及红外光谱分析和蛋白质含量测定表明该多糖为蛋白多糖,其蛋白质和多糖含量在水提多糖中分别为１３％和６２％、在酸提多糖中分别为５％和９１％。在碱提多糖中分别为３％和８１％。组成该多糖的主要单糖是葡萄糖和木糖。     

深层发酵香菇水溶性胞外多糖的生物学活性

由香菇ＣＬ－２菌丝发酵上清液中分离到水溶性胞外多糖（ＨＥＰ）。研究表明,ＨＥＰ具有较强的免疫增效作用。它能有效促进正常小鼠腹腔巨噬细胞的吞噬功能,显著提高Ｔ淋巴细胞的百分含量,对小鼠体液免疫也有促进作用。ＨＥＰ对肉瘤Ｓ１８０的抑制率达３９．７％,并能显著延长荷瘤（ＥＡＣ）小鼠的存活时间,延长率达４０．５％。ＨＥＰ对牛艾滋病毒（ＢＩＶ）有直接抑制作用,抑制率为６６．７％。     

枸杞子糖蛋白的分离纯化、物化性质及糖肽键特征

从宁夏枸杞子提取得到的粗多糖,经ＤＥＡＥ－Ｃｅｌｌｕｌｏｓｅ和ＳｅｐｈａｄｅｘＧ－１００柱层析,得到均一的枸杞子糖蛋白ＬｂＧＰ。分子量由ＳＤＳ－ＰＡＧＥ测定为８８ｋｄ,糖含量为７０％,糖组成为Ａｒａ：Ｇａｌ：Ｇｌｃ＝２．５：１．０：１．０（摩尔比）,并含有其他１８种天然氨基酸。初步分析表明ＬｂＧＰ是Ｏ－连接的糖蛋白。     

Conformational mapping of the N-terminal peptide of HIV-1 gp41 in lipid detergent and aqueous environments using 13C-enhanced Fourier transform infrared spectroscopy

The N-terminal domain of HIV-1 glycoprotein 41,000 (gp41) participates in viral fusion processes. Here, we use physical and computational methodologies to examine the secondary structure of a peptide based on the N terminus (FP; residues 1-23) in aqueous and detergent environments. (12)C-Fourier transform infrared (FTIR) spectroscopy indicated greater alpha-helix for FP in lipid-detergent sodium dodecyl sulfate (SDS) and aqueous phosphate-buffered saline (PBS) than in only PBS. (12)C-FTIR spectra also showed disordered FP conformations in these two environments, along with substantial beta-structure for FP alone in PBS. In experiments that map conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using FP peptides labeled with (13)C-carbonyl. (13)C-FTIR results on FP in SDS at low peptide loading indicated alpha-helix (residues 5 to 16) and disordered conformations (residues 1-4). Because earlier (13)C-FTIR analysis of FP in lipid bilayers demonstrated alpha-helix for residues 1-16 at low peptide loading, the FP structure in SDS micelles only approximates that found for FP with membranes. Molecular dynamics simulations of FP in an explicit SDS micelle indicate that the fraying of the first three to four residues may be due to the FP helix moving to one end of the micelle. In PBS alone, however, electron microscopy of FP showed large fibrils, while (13)C-FTIR spectra demonstrated antiparallel beta-sheet for FP (residues 1-12), analogous to that reported for amyloid peptides. Because FP and amyloid peptides each exhibit plaque formation, alpha-helix to beta-sheet interconversion, and membrane fusion activity, amyloid and N-terminal gp41 peptides may belong to the same superfamily of proteins.     

真菌多糖抗氧化活性的研究进展

真菌多糖的抗氧化作用目前正成为国内外众多学科领域研究的热点之一,对真菌子实体多糖、菌丝体多糖和胞外多糖的抗氧化活性研究现状进行了综述,并对其研究前景进行了展望,旨在为真菌多糖的进一步研究和利用提供参考.     

Structural and functional properties of peptides based on the N-terminus of HIV-1 gp41 and the C-terminus of the amyloid-beta protein

Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26-42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer's. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26-42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26-42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26-42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’).     

Sensitive fluorescence polarization technique for rapid screening of alpha-synuclein oligomerization/fibrillization inhibitors

Parkinson's disease (PD) is characterized by the accumulation of fibrillar alpha-synuclein (alpha-Syn) inclusions known as Lewy bodies (LBs) and Lewy neurites. Mutations in the alpha-Syn gene or extra copies thereof cause familial PD or dementia with LBs (DLB) in rare kindreds, but abnormal accumulations of wildtype alpha-Syn also are implicated in the pathogenesis of sporadic PD, the most common movement disorder. Insights into mechanisms underlying alpha-Syn mediated neurodegeneration link alpha-Syn oligomerization and fibrillization to the onset and progression of PD. Thus, inhibiting alpha-Syn oligomer or fibril formation is a compelling target for discovering disease modifying therapies for PD, DLB, and related synucleinopathies. Although amyloid dyes recognize alpha-Syn fibrils, efficient detection of soluble oligomers remains a challenge. Here, we report a novel fluorescence polarization (FP) technique for examining alpha-Syn assembly by monitoring changes in its relative molecular mass during progression of normal alpha-Syn from highly soluble monomers to higher order multimers and thence insoluble amyloid fibrils. We report that FP is more sensitive than conventional amyloid dye methods for the quantification of mature fibrils, and that FP is capable of detecting oligomeric alpha-Syn, allowing for rapid automated screening of potential inhibitors of alpha-Syn oligomerization and fibrillization. Furthermore, FP can be combined with an amyloid dye in a single assay that simultaneously provides two independent biophysical readouts for monitoring alpha-Syn fibrillization. Thus, this FP method holds potential to accelerate discovery of disease modifying therapies for LB PD, DLB, and related neurodegenerative synucleinopathies.     

Common deletion of SMAD4 in juvenile polyposis is a mutational hotspot

Juvenile polyposis (JP) is an autosomal dominant syndrome in which affected patients develop upper- and/or lower-gastrointestinal (GI) polyps. A subset of families with JP have germline mutations in the SMAD4 (MADH4) gene and are at increased risk of GI cancers. To date, six families with JP have been described as having the same SMAD4 deletion (1244-1247delAGAC). The objective of the present study is to determine whether this deletion is a common ancestral mutation or a mutational hotspot. DNA from members of four families with JP, from Iowa, Mississippi, Texas, and Finland, that had this 4-bp deletion was used to genotype 15 simple tandem repeat polymorphism (STRP) markers flanking the SMAD4 gene, including 2 new STRPs within 6.3 and 70.9 kb of the deletion. Haplotypes cosegregating with JP in each family were constructed, and the distances of the closest markers were determined from the draft sequence of the human genome. No common haplotype was observed in these four families with JP. A 14-bp region containing the deletion had four direct repeats and one inverted repeat. Because no common ancestor was suggested by haplotype analysis and the sequence flanking the deletion contains repeats frequently associated with microdeletions, this common SMAD4 deletion in JP most likely represents a mutational hotspot.     

Effect of hamster pregnancy on female protein, a homolog of serum amyloid P component.

Pentraxins such as human serum amyloid P component (SAP) and C reactive protein (CRP) represent an ancient family of proteins that are ubiquitous in nature and have evolved with little change in structure or regulation. The pentraxin in the Syrian hamster (Mesocricetus auratus) is unique because it is preferentially expressed in the female at high constitutive levels and accordingly called female protein (FP) or FP(SAP) due to its close homology with human SAP. The high levels of FP in female serum (100-fold greater than male serum) suggested its role in hamster pregnancy, one of the shortest of any eutherian mammal. We determined the serum FP concentration in pregnant Syrian hamsters and found a marked decrease (>80%) at term with the nadir at parturition with subsequent increase. A similar downregulation of FP was found in the normal female Syrian hamster after injury (acute phase response), so in both cases the assumed beneficial effects were achieved with less, rather than more pentraxin, a paradoxical pentraxin response. The fall in serum FP concentration could represent a response to protect the fetus from the high and potentially toxic level of FP normally found in the female, that is harmful because of its association with amyloidosis. An FP that is 97.5% identical to Syrian hamster FP is found in the Turkish hamster (Mesocricetus brandti), although serum levels in females are much lower, and amyloid is very rare. During pregnancy/parturition of Turkish hamsters, the serum level of FP remained remarkably constant. In a more distantly related hamster, the Armenian hamster (Cricetulus migratorius), serum FP actually increased during pregnancy and at parturition in a manner similar to that found in the Armenian hamster during an acute phase response. The heterogeneity of FP kinetics during pregnancy in these three species of hamster indicates pleomorphic gene structure for regulation of their similar FPs, and suggests that this protein may have a different function in the pregnancy of each species.     

Biochemical characterization and site-directed mutational analysis of the double chitin-binding domain from chitinase 92 of Aeromonas hydrophila JP101

Chitinase 92 from Aeromonas hydrophila JP101 contains C-terminal repeated chitin-binding domains (ChBDs) which were named ChBD(CI) and ChBD(CII) and classified into family 5 carbohydrate-binding modules on the basis of sequence. In this work, we constructed single and double ChBD by use of the pET system, which expressed as isolated ChBD(CII) or ChBD(CICII). Polysaccharide-binding studies revealed that ChBD(CICII) not only bound to chitin, but also to other insoluble polysaccharides such as cellulose (Avicel) and xylan. In comparison with ChBD(CII), the binding affinities of ChBD(CICII) are about 10- and 12-fold greater toward colloidal and powdered chitin, indicating that a cooperative interaction exists between ChBD(CI) and ChBD(CII). In order to investigate the roles of the highly conserved aromatic amino acids in the interaction of ChBD(CICII) and chitin, we have performed site-directed mutagenesis. The data showed that W773A, W792A, Y796A and W797A mutant proteins exhibited a much weaker affinity for chitin than wild-type protein, suggesting that these residues play important roles in chitin binding.     

Amyloid peptides derived from CsgA and FapC modify the viscoelastic properties of biofilm model matrices

The bacterial biofilm is a complex environment of cells, which secrete a matrix made of various components, mainly polysaccharides and proteins. An understanding of the precise role of these components in the stability and dynamics of biofilm architecture would be a great advantage for the improvement of anti-biofilm strategies. Here, artificial biofilm matrices made of polysaccharides and auto-assembled peptides were designed, and the influence of bacterial amyloid proteins on the mechanical properties of the biofilm matrix was studied. The model polysaccharides methylcellulose and alginate and peptides derived from the amyloid proteins curli and FapC found in biofilms of Enterobacteriaceae and Pseudomonas, respectively, were used. Rheological measurements showed that the amyloid peptides do not prevent the gelation of the polysaccharides but influence deformation of the matrices under shear stress and modify the gel elastic response. Hence the secretion of amyloids could be for the biofilm a way of adapting to environmental changes.     

Hormonal regulation of JP29 in the epidermis during larval development and metamorphosis in the tobacco hornworm,Manduca sexta

Previous studies have shown that the larval epidermis of the tobacco hornworm, Manduca sexta, contains a 29 kDa nuclear protein (JP29) that binds pothoaffinity analogs of juvenile hormone (JH), but does not bind JH I with high affinity. We now find that JP29 is also associated with the insecticyanin granules, and we show that JP29 mRNA is regulated in a complex fashion by both 20-hydroxyecdysone (20E) and JH. Studies with day 2 fourth instar larval epidermis in vitro showed that a molting concentration 12 μg/ml) of 20E caused the disappearance of JP29 mRNA, irrespective of the presence or absence of JH; this effect was dependent on the concentration of 20E (ED₅₀=200 ng/ml). The reappearance of JP29 mRNA around the time of ecdysis required the presence of JH at head capsule slippage (HCS), since little appeared in larvae allatectomized about 6 h before HCS unless JH I was applied at the time of HCS. Maintenance of JP29 mRNA in fifth instar epidermis also required the continued presence of JH in both isolated abdomens and in vitro. Culture of either day 1 or day 2 fifth instar epidermis without hormones for 24 h caused decline of JP29 mRNA, which was accelerated by 20E in a concentration-dependent manner (ED₅₀ = 30 and 10 ng/ml 20E respectively). When day 2 epidermis was exposed to 500 ng/ml 20E for 24 h to cause pupal commitment, JP29 mRNA disappeared. Neither methoprene nor JH I (in either the presence or the absence of the esterase inhibitor O-ethyl, S-phenyl phosphamidethiolate [EPPAT]) was able to prevent this loss, although both slowed its rate. The mRNA for the larval cuticle protein LCP14 was found to be regulated similarly to that for JP29 by 20E, but differently by JH. The JP29 protein was relatively long-live, persisting after the disappearance of its mRNA for at least 19 h during the larval molt and for more than 24 h in vitro. Although trace amounts of JP29 are found for the first 12 h after pupal ecdysis, injection of 5 μg JH II into pupae during the critical period to cause the synthesis of a second pupal cuticle had no effect on the amount of JP29 present. Thus, although the presence of JP29 in larval epidermis is associated with and dependent on JH, high amounts are not associated with the “status quo” action of JH on the pupa. The role of this protein consequently remains obscure. Arch. Insect Biochem. Physiol. 34:409–428, 1997. © 1997 Wiley-Liss, Inc.     

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson’s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.