Rapid analysis of mono-saccharides and oligo-saccharides in hydrolysates of lignocellulosic biomass by HPLC

HPLC using pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) was used to analyse mono-saccharides and oligo-saccharides in hydrolysates of lignocellulosic biomass. PMP derivatives, including those of mannose, rhamnose, cellobiose, glucose, xylose and arabinose, were separated within 14 min with detection at 254 nm. The method was also suitable for xylo-oligosaccharides (XOS): PMP derivatives of xylohexaose, xylopentaose, xylotetraose, xylotriose and xylobiose were well separated under the same conditions. The method was used to determine the mono-saccharide composition of Miscanthus and evaluate the production of XOS from enzymatic hydrolysis of crude xylan.     

Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography

Counter-current chromatography (CCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate-water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, 1HNMR and 13CNMR.     

SSR based genetic diversity of pigmented and aromatic rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes of the western Himalayan region of India

A set of 24 of SSR markers were used to estimate the genetic diversity in 16 rice genotypes found in Western Himalayas of Kashmir and Himachal Pradesh, India. The level of polymorphism among the genotypes of rice was evaluated from the number of alleles and PIC value for each of the 24 SSR loci. A total of 68 alleles were detected across the 16 genotypes through the use of these 24 SSR markers The number of alleles per locus generated varied from 2 (RM 338, RM 452, RM 171) to 6 (RM 585, RM 249, RM 481, RM 162). The PIC values varied from 0.36 (RM 1) to 0.86 (RM 249) with an average of 0.62 per locus. Based on information generated, the genotypes got separated in six different clusters. Cluster 1 comprised of 4 genotypes viz; Zag 1, Zag 13, Pusa sugandh 3, and Zag 14, separated from each other at a similarity value of 0.40. Cluster second comprised of 3 landraces viz; Zag 2. Zag 4 and Zag10 separated from each other at a similarity value of 0.45. Cluster third comprised of 3 genotypes viz; Grey rice, Mushk budji and Kamad separated from each other at a similarity value of 0.46. Cluster fourth had 2 landraces viz; Kawa kreed and Loual anzul, and was not sub clustered. Fifth cluster had 3 genotypes viz; Zag 12, Purple rice and Jhelum separated from each other at a similarity value of 0.28. Cluster 6 comprised of a single popular variety i.e. Shalimar rice 1 with independent lineage.     

大白鼠妊娠早期(1—6天)子宫孕酮受体的变化

本实验中大鼠妊娠第三天(D_3)出现血浆孕酮含量和子宫细胞胞核中孕酮受体含量显著同步升高和胞质中孕酮受体含量明显下降的现象,为D_5胚泡着床准备了必要的条件。D_6时血浆孕酮,胞质和胞核中孕酮受体以及子宫重量均升高,标志胚泡着床后的生理变化。     

Rapid determination of ethylene glycol at toxic levels in serum and urine

The rapid gas chromatographic detection and determination of ethylene glycol in biological fluids is described. Phenylboronic acid in acetone was used for the esterification of glycol. The phenylboronates of ethylene glycol and 1,2-propylene glycol are not separated on a packed column of medium polarity (OV-17), but they can be separated on a non-polar column (OV-101). In both instances 1,3-propylene glycol can be used as an internal standard. The method requires only 100 μl of serum or urine and is suitable for trace analysis in an emergency toxicological laboratory. The utility of the method is demonstrated on two cases of human intoxication with ethylene glycol.     

超滤膜纯化绞股蓝皂苷的工艺

探讨采用超滤膜分离纯化绞股蓝总皂苷的工艺。以固形物得率和绞股蓝总皂苷含量为评价指标进行正交试验,对操作压力、溶液温度和膜的规格等因素进行优选。发现最佳工艺条件为选择截留相对分子质量为3×104的超滤膜,在溶液温度40℃、操作压力2.0×106Pa条件下纯化效果较好;所选择的超滤膜纯化绞股蓝皂苷的工艺操作简单可靠,所得产品纯度高。     

Relative order determination of four Yp cosmids on metaphase and interphase chromosomes by two-color competitive in situ hybridization

Summary Two-color competitive in situ hybridization was used to cytogenetically order four Yp cosmid probes, located in the pseudo-autosomal and TDF regions. The probes were hybridized by pairs to metaphase and interphase chromosomes. On metaphase chromosomes, determination of order between sequences separated by 3 Mb from each other was possible on a statistical basis, whereas the relative position of sequences 0.6Mb apart could not be determined. On interphase chromosomes the complete order between sequences separated by 0.6– 6Mb was obtained rapidly by measuring the distances between two cosmid spots of every cosmid pair used in 28 to 60 nuclei. Results demonstrate the potential power of fluorescent in situ hybridization at interphase for high resolution cosmid mapping.     

Chiral separation of T3 enantiomers using stereoselective antibodies as a selector in micro-HPLC

This work deals with the application of stereoselective antibodies against L-T3 as a tailor-made chiral selector in micro-HPLC. The separations were performed in microbore columns using commercially available anti-L-T3 antibodies chemically bonded to 5 microm silica gel. The enantiomers of T3 were baseline separated under mild continuous isocratic elution conditions using 10 mM phosphate buffer, pH 7.4. The D-enantiomer eluted with the void volume, while the L-enantiomer was retained by the antibody phase and eluted second. An indirect competitive and non-competitive enzyme linked immunosorbent assay (ELISA) was used for testing the stereoselectivity of anti-L-T3 antibodies.     

Stress-Induced Visceral Hypersensitivity in Maternally Separated Rats Can Be Reversed by Peripherally Restricted Histamine-1-Receptor Antagonists

Background

The histamine-1 receptor (H1R) antagonist ketotifen increased the threshold of discomfort in hypersensitive IBS patients. The use of peripherally restricted and more selective H1R antagonists may further improve treatment possibilities. We examined the use of fexofenadine and ebastine to reverse post-stress visceral hypersensitivity in maternally separated rats.

Methods

The visceromotor response to colonic distension was assessed in adult maternally separated and nonhandled rats pre- and 24 hours post water avoidance. Subsequently rats were treated with vehicle alone or different dosages of fexofenadine (1.8 and 18 mg/kg) or ebastine (0.1 and 1.0 mg/kg) and re-evaluated. Colonic tissue was collected to assess relative RMCP-2 and occludin expression levels by Western blot and histamine-1 receptor by RT-qPCR. β-hexosaminidase release by RBL-2H3 cells was used to establish possible mast cell stabilizing properties of the antagonists.

Key results

Water avoidance only induced enhanced response to distension in maternally separated rats. This response was reversed by 1.8 and 18 mg/kg fexofenadine. Reversal was also obtained by 1.0 but not 0.1 mg/kg ebastine. RMCP-2 expression levels were comparable in these two ebastine treatment groups but occludin was significantly higher in 1.0 mg/kg treated rats. There were no differences in histamine-1 receptor expression between nonhandled and maternally separated rats. Fexofenadine but not ebastine showed mast cell stabilizing quality.

Conclusions

Our results indicate that the peripherally restricted 2^nd generation H1-receptor antagonists fexofenadine and ebastine are capable of reversing post stress visceral hypersensitivity in rat. These data justify future IBS patient trials with these well tolerated compounds.     

A 32P postlabeling assay for determining the incorporation of bromodeoxyuridine into cellular DNA

Randerath's procedure for 32P postlabeling of 3'-monophosphate deoxyribonucleotides from digests of cellular DNA has been modified. 3'-Monophosphate deoxyribonucleotides are converted to 3',5'-bis[32P]phosphate deoxyribonucleotides with polynucleotide kinase and [32P]ATP; these products are enzymatically converted by P1 nuclease and polynucleotide kinase into 5'-[32P]monophosphate deoxyribonucleotides, which are separated from [32P]ATP on an anion-exchange column eluted with 0.1 M NaH2PO4, pH 6.5. Labeled mononucleotides in the effluent are separated by high-performance liquid chromatography. Values for the base composition of calf thymus DNA determined with this modified assay compare very favorably with reported values. The assay was used to measure the level of incorporation of the clinically useful agent bromodeoxyuridine into the DNA of 9L rat brain tumor cells. The modified assay appears to be a very accurate method for the determination of levels of base analogs incorporated into DNA.     

Restriction fragment fingerprint and genome sizes of Staphylococcus species using pulsed-field gel electrophoresis and infrequent cleaving enzymes.

Large restriction fragments of genomic DNA from Staphylococcus species were separated by pulsed-field gel electrophoresis (PFGE). Five different strains of S. aureus (ISP8, SAU3A, PS96, ATCC 6538, ATCC 15564) and three representative strains of S. haemolyticus SM102, S. warneri MCS4, S. cohnii LK478 from human hosts, and one strain of S. aureus (ATCC 8432) from an avian host were used in this study. Since Staphylococcus is A + T rich (approximately 67%), restriction fragments were obtained by digesting chromosomal DNA with endonucleases that recognize GC-rich sequences. Five enzymes Csp I, Sma I, Ecl XI, Ksp I, or Sac II were used for generation of few (7 to 16) distinctly separated fragments, with average sizes in the range of 200-300 kb. The size distribution of restriction fragments for each enzyme for each strain produced a strain-identifying fingerprint, and the genome size of each strain was determined from such restriction fragments separated by PFGE.     

A high-performance liquid chromatographic method for the determination of dolichyl phosphates in tissues

A sensitive high-performance liquid chromatographic method has been developed for the determination of dolichyl phosphate homologs in tissues. Dolichyl phosphates were methylated and derivatized with 3-(9-anthryl)-diazo-2-propene to yield methyl 3-(9-anthryl)-2-propenyl dolichyl phosphates. As an internal standard, 2,2-didecaprenylethyl phosphate was used. The derivatives were separated on a reversed-phase column using isocratic elution, and detected fluorometrically. The method with fluorescence detection was sufficiently sensitive to measure the concentration of dolichyl phosphate homologs in tissues.     

高效柴油降解菌Acinetobacter sp.W3分离鉴定及降解酶基因扩增分析

从柴油污染的海水样品中分离高效柴油降解细菌,分析菌株对柴油的降解能力及降解酶基因,为海洋柴油污染的生物修复奠定基础。选取浙江定海港柴油污染的海水样品,进行降解菌的富集培养;采用常规方法分离筛选高效柴油降解菌。利用革兰氏染色、形态学观察、生理生化鉴定及16S rDNA分析等方法对降解菌株进行种属鉴定。采用紫外吸收法测定菌株对柴油的降解率。采用PCR方法、核酸序列测定和比对,对其降解酶基因进行扩增分析。筛选出一株高效降解菌,形态学观察及生理生化鉴定初步确定为不动杆菌。16S rDNA序列分析及比对结果表明,其16S rDNA序列与威尼斯不动杆菌(Acinetobacter venetianus)属的序列同源性达到99.7%,命名为不动杆菌W3(Acinetobactersp.W3),该菌对柴油的7 d降解率达到84.7%。PCR方法从Acinetobactersp.W3菌株中的基因组DNA和质粒DNA上扩增到了大小为540 bp的烷烃羟化酶基因alkB和864 bp的CYP153A部分DNA片段,分别与Acinetobacter venetianus1-D-2的alkB和Acinetobactersp.OC4、Acinetobactersp.EB104的CYP153具有99%和98%的同源性。从定海港口柴油污染海水分离得到一株高效柴油降解菌Acinetobactersp.W3,该菌属于不动杆菌属,含有烷烃降解酶基因,能高效降解柴油污染物,有望应用于海水柴油污染的生物修复。     

Automated protein precipitation by filtration in the 96-well format

The use of automated protein precipitation by filtration in the 96-well format as a rapid sample preparation technique for high throughput bioanalysis using liquid chromatography tandem mass spectrometry is reported. A robotic sample processor is used to aspirate sequentially a plasma sample and acetonitrile separated by air gaps. These are then mixed by being dispensed into individual channels of a 96-well filter block. The resulting supernatant is separated from the precipitated plasma proteins by the application of gentle vacuum using a custom manifold. The filtered supernatants are collected into a deep well microtitre plate, evaporated to dryness using a heated 96-well dry down station and reconstituted in water prior to analysis. The efficiency of the extraction procedure is measured by the Lowry method for determining protein concentration. This method was used to optimise both the volume and the order of reagent addition, and to compare several prototype 96-well filter blocks. Using the optimised procedure a specific, precise and accurate method was developed for the β-agonist salbutamol in rabbit plasma with a calibration range of 1 to 100 ng/ml from 100 μl of sample.     

Quantitation of the glycation intermediate 3-deoxyglucosone by oxidation with rabbit liver oxoaldehyde dehydrogenase to 2-keto-3-deoxygluconic acid followed by high-performance liquid chromatography

A simple and sensitive method for the detection of 3-deoxyglucosone was developed using oxidation with crude oxoaldehyde dehydrogenase to 2-keto-3-deoxygluconic acid followed by high-performance liquid chromatography (HPLC). Oxoaldehyde dehydrogenase was prepared from rabbit liver and partially characterized. 2-Keto-3-deoxygluconic acid produced from 3-deoxyglucosone by oxoaldehyde dehydrogenase was derivatized with 1,2-diamino-4,5-methylenedioxybenzene, and the fluorescent products were detected and quantitated by HPLC using a solvent containing borate. In the presence of borate, 2-keto-3-deoxygluconic acid was completely separated from N-acetylneuraminic acid. The detection limit of 3-deoxyglucosone was 2.5 pmol/injection (10 μl) at a signal-to-noise ratio of 3. This method was used to confirm the inhibitory effect of aminoguanidine on glycation.     

蛋白质双向电泳、印迹转移及蛋白质合成无细胞体系技术在小鼠腹水细胞EF-3研究中的应用

本文应用近年发展起来的生化技术――蛋白质双向电泳（其第一向为等电聚焦,第二向为SDS凝胶电泳）,将小鼠腹水细胞核糖体蛋进行了指纹分离。并利用蛋白质印迹转移（Western blotting）,将转移后的硝酸纤维膜与交联了碱性磷酸酶的第二抗体和抗酵母EF-3抗体反应,证实该核糖体蛋白含有EF-3同源片段,进而制备了蛋白质合成无细胞体系。通过测定PolyU指导下3 H-phe掺入活力的免疫失活实验,初步证实此同源片段是小鼠腹水细胞蛋白质合成所必需。 The ribosomal proteins of H22a cell,were separated with the method of two-D gel electrophoresis (the first dimention is isoelectric focus and the second is SDS PAGE).Then the Western blotting was used,the transferred nitrocellulose sheet was treated with antiyeast EF-3 antibody and the second antibody bonded with alklinephosphoesterase.The result shows that the ribosomal proteins have a homologous fragment to yeast EF-3 factor.The cell-free system of protein synthesis was also established.By determing the activity of polyU direeted 3H-phe intervention in immunodeactivitive experiment,it is primaril confirmed that this fragment is the esscntial for the protein biosynthesis in H22a cell.     

纳豆芽孢杆菌Bna05菌株产抗霉脂肽的鉴定

【目的】分析枯草芽孢杆菌纳豆菌亚种Bna05菌株代谢产物中脂肽类物质的存在情况,并探讨它们在抗霉功能中所发挥的作用。【方法】利用特异性引物对Bna05菌株进行脂肽合成酶类基因片段扩增、测序和BLAST比对分析;通过平板抑菌圈区域取样法获得Bna05菌株的高抗霉活性代谢产物,对该产物进行反相高效液相色谱(RP-HPLC)分离;用琼脂微稀释法测定分离物的抗霉活性,并对活性分离物进行质谱鉴定。【结果】Bna05菌株含有sfp和srf AA基因,未检测到itu C、itu D、fen D、fen ACE、bym B、bym C基因;RP-HPLC分离得到3组抗霉活性物质F_2、F_3和F_4,F_2中未检测到脂肽类物质,从F_3和F_4中分别鉴定出两类Surfactin同系物:V_7-surfactin和I/L_7-surfactin。两类Surfactin分别与F_2组合使用时,均表现出抗霉协同作用;此外,与Surfactin单独使用相比,两类Surfactin混合物与F_2组合后的协同抗霉活性得到进一步增强。【结论】Bna05菌株所产脂肽类物质主要是V_7-surfactin和I/L_7-surfactin,Surfactin与Bna05菌株所产其它活性物质之间存在抗霉协同作用,而V_7-surfactin和I/L_7-surfactin的同时存在,对于增强这种协同抗霉作用是有利的。     

Ucon-benzoyl dextran aqueous two-phase systems: protein purification with phase component recycling

Benzoyl dextran with a degree of substitution of 0.18 was synthesized by reacting dextran T500 with benzoyl chloride. A new type of aqueous two-phase system composed of benzoyl dextran as bottom phase polymer and the random copolymer of ethylene oxide and propylene oxide (Ucon 50-HB-5100) as top phase polymer has been formed. The phase diagram for the system Ucon 50-HB-5100-benzoyl dextran with a degree of substitution of 0.18 was determined at room temperature. This two-phase system has been used to purify 3-phosphoglycerate kinase from bakers' yeast. The top-phase polymer (Ucon) can be separated from target enzyme by increasing the temperature. The bottom-phase polymer (benzyol dextran) could be recovered by addition of salt. Yeast homogenate was partitioned in a primary Ucon 50-HB-5100-benzoyl dextran aqueous two-phase system. After phase separation the top phase was removed and temperature-induced phase separation was used for formation of a water phase and a Ucon-rich phase. The benzoyl dextran-enriched bottom phase from the primary system was diluted, and the polymer was separated from water by addition of Na₂SO₄.     

Internal fluorescence labeling with fluorescent deoxynucleotides in two-label peak-height encoded DNA sequencing by capillary electrophoresis.

Fluorescently labeled deoxynucleotides were used for internal labeling of DNA sequencing fragments generated in a two-color peak-height encoded protocol. Sequenase and a manganese-containing buffer were used to generate uniform peak heights. Tetramethyl rhodamine - dATP was used in a labeling step, followed by termination with ddATP and ddCTP in a 3:1 ratio. Fluorescein - dATP was used in a second reaction, followed by termination with ddGTP and ddTTP in a 3:1 ratio. The fragments were pooled and separated by capillary gel electrophoresis. The results were compared with peak-height encoded sequencing based on fluorescently labeled primers. The dye-labeled primers produced higher resolution separations for shorter fragments. However, dye-labeled primer fragments suffered from an earlier onset of biased reptation and produced shorter sequencing reads. Fragments from 50 to at least 500 bases in length could be sequenced with the internal labels.     

Species delimitation in <Emphasis Type="Italic">Epilobium</Emphasis> (sec. <Emphasis Type="Italic">Epilobium</Emphasis>, Onagraceae): morphological,molecular and palynological data

The genus Epilobium L. (Family Onagraceae) with about 200 species is taxonomically complicated due to wide inter-specific gene flow. There is no report on biosystematics of Epilobium species in Iran, therefore we studied 5 species of this genus using a combination of morphological, molecular (ISSR) and palynological data to delimit these species. All data revealed high degree of morphological and genetic overlap among the studied species. However, they could be separated when all studied features were used together. Morphological characters could delimit 3 species while genetic data separated them in two major groups. However, palynological data could delimit all five species studied. The species relationship was highly compatible in all three data sources used.