Purification of a synthetic oligonucleotide by anion exchange chromatography: method optimisation and scale-up

A single-step chromatographic method for purification of a synthetic 20-mer oligonucleotide is described. Method optimisation was conducted at laboratory scale where 30 mg crude sample was purified per run with a yield of 17 mg pure oligonucleotide. The protocol was scaled-up in steps to achieve 5-, 58- and a final 230-fold scale-up. At the final scale, 7.0 g of crude material was purified with a yield of 4.1 g product. The purity of the oligonucleotide was in all scales higher than 97%. The cycle time was 110 min, which corresponds to a purification capacity of about 90 g crude oligonucleotide material per 24 h.     

Specific method for determination of gefitinib in human plasma, mouse plasma and tissues using high performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using liquid chromatography-tandem mass spectrometry (LC/MS/MS) for determination of gefitinib in human plasma and mouse plasma and tissue. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma or a 200 mg/mL tissue homogenate diluted 1/10 in human plasma with 0.3 mL acetonitrile. Separation of the compounds of interest, including the internal standard (d8)-gefitinib, was achieved on a Waters X-Terra C18 (50 mm x 2.1 mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile-water (70:30, v/v) containing 0.1% formic acid and isocratic flow at 0.15 mL/min for 3 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 1-1000 ng/mL for the human plasma samples and 5-1000 ng/mL for mouse plasma and tissue samples with values for the coefficient of determination of > 0.99. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (< 15%). This method was subsequently used to measure concentrations of gefitinib in mice following administration of a single dose of 150 mg/kg intraperitoneally and in cancer patients receiving an oral daily dose of 250 mg.     

Measurement of muramic acid in marine sediments by high performance liquid chromatography

Bacterial biomass in marine sediments may be estimated from the amount of muramic acid present. A method for determining muramic acid by high performance liquid chromatography is described, which is simpler and faster than other methods. Muramic acid is released from sediment by acid hydrolysis, and assayed as an o-phthaldialdehyde derivative.     

Determination of L- and D-amino acids in foodstuffs by coupling of high-performance liquid chromatography with enzyme reactors

Summary. A technique is described for the enantiomeric determination of L- and D-amino acids. It works on the principle that the separation efficiency of high-performance liquid chromatography is coupled with the specificity of enzymes and the sensitivity of electrochemical detection. After separation on a lithium cation-exchange column the amino acids are converted into keto acids and hydrogen peroxide under catalyzation of L- or D-amino acid oxidase. Hydrogen peroxide is detected amperometrically. The method has been tested by the analysis of beer, port, sherry, wine and fruit juice. A main emphasis was put onto the determination of D-alanine which can serve as an indicator for bacterial contamination. It is shown that a coupling of HPLC with enzyme reactors is a suitable technique for the rapid detection of this marker. Received April 14, 1999, Accepted September 15, 1999     

A unique example of a pseudo-peptide containing noncoded amino acids self-assembling into a supramolecular β-sheet-like structure in crystals

Summary The crystal structure of a pseudo-peptide with noncoded amino acids (such as N-substituted 3-aminophenylacetic acid and α-aminoisobutyric acid) exhibits almost and extended backbone conformation and this pseudo-peptide self-assembled to form an infinite hydrogen bonded, supramolecular, antiparallel β-sheet-like structure in solid state.     

Biochemical changes induced by accelerated ageing in Bambusa bambos seeds

Decrease in seed viability and germination rate may be caused by biochemical changes associated with seed ageing. Different biochemical assays were conducted to investigate the changes occurring at the ageing of Bambusa bambos seeds. A reduction in the total content of food reserves such as sugars, proteins and lipids were recorded. Decreased activity of peroxidase, acid phosphatase, alkaline phosphatase were also noticed during accelerated ageing. A substantial increase in total free amino acids and the activity of amylases confirms the degradation of stored biomolecules in seeds during ageing. This revised version was published online in August 2006 with corrections to the Cover Date.     

高效液相色谱法测定必特螺旋霉素发酵液中有机酸

必特螺旋霉素是运用基因工程技术获得的工程茵产生的一组以异戊酰螺旋霉素为主要成分的多组分新型抗生素,其前体为乙酸、丙酸、丁酸和异戊酸等有机酸。本研究利用高效液相色谱法以0．01mol／L磷酸缓冲液（pH2．3）和甲醇为流动相,分别在反相C8（α-酮戊二酸、乙酸、柠檬酸、琥珀酸、丙酸）和C18（丁酸、异戊酸）柱上定量测定了必特螺旋霉素前体酸和三羧酸循环相关有机酸。所建立的测定方法的相对标准偏差为0．10％～0．42％,回收率为93．19％～102．08％。     

Expression of bacterial hemoglobin genes to improve astaxanthin production in a methanotrophic bacterium Methylomonas sp

Astaxanthin has been widely used as a feed supplement in poultry and aquaculture industries. One challenge for astaxanthin production in bacteria is the low percentage of astaxanthin in the total carotenoids. An obligate methanotrophic bacterium Methylomonas sp. 16a was engineered to produce astaxanthin. Astaxanthin production appeared to be dramatically affected by oxygen availability. We examined whether astaxanthin production in Methylomonas could be improved by metabolic engineering through expression of bacterial hemoglobins. Three hemoglobin genes were identified in the genome of Methylomonas sp. 16a. Two of them, thbN1 and thbN2, belong to the family of group I truncated hemoglobins. The third one, thbO, belongs to the group II truncated hemoglobins. Heterologous expression of the truncated hemoglobins in Escherichia coli improved cell growth under microaerobic conditions by increasing final cell densities. Co-expression of the hemoglobin genes along with the crtWZ genes encoding astaxanthin synthesis enzymes in Methylomonas showed higher astaxanthin production than expression of the crtWZ genes alone on multicopy plasmids. The hemoglobins likely improved the activity of the oxygen-requiring CrtWZ enzymes for astaxanthin conversion. A plasmid-free production strain was constructed by integrating the thbN1–crtWZ cassette into the chromosome of an astaxanthin-producing Methylomonas strain. It showed higher astaxanthin production than the parent strain.     

Simulation of formation of α-helices and β-hairpins in water-soluble proteins by the code-based physics method

One of the possible ways for a complete and final decision of the problem of the determination of three-dimensional structure of proteins from their amino acid sequence is simulation of protein three-dimensional structure formation. For the performance of this task it is suggested to use the code-based physics method developed by the author. In this article a simulation of the α-helix and β-hairpin formation in water-soluble proteins as a start of the realization of this plan is described. Results of the simulation are compared from experimental data for 14 proteins of no more than 50 amino acids and, therefore, with a small number of α-helices and β-strands (to meet limits of simulation process) and secondary structure predictions by the best current methods of protein secondary structure prediction, PSIpred, PORTER and PROFsec. The secondary structure of proteins, obtained as a result of the simulation of α-helix and β-hairpin formation by the code-based physics method, agrees completely with the experiment, while the secondary structure predicted by the PSIpred, PORTER, and PROFsec methods contains significant differences from the experimental data.     

Immunohistological Analysis of Chemically Induced Proteins in Sugar Beet

Tissue-specific distribution of basic β-1,3-glucanase (Glu2), basic class II chitinase (Ch2), basic class IV chitinase (Ch4), and acidic class III chitinase (SE2) were examined both in leaves and roots of sugar beet treated with salicylic acid (SA), benzothiadiazole (BTH) and glycine betaine. Protein localization was monitored by immunohistological analysis using specific antibodies. BTH, SA as well as glycine betaine induced both Glu2 and chitinase isozymes in leaves and roots of treated plants. The enzymes were accumulated in extracellular space and cell walls. They were mostly deposited in parenchyma cells of leaves and cortex parenchyma and endodermis of roots. In leaf tissues, BTH and SA induced proteins more effectively than glycine betaine but the effect of glycine betaine in roots was as efficient as BTH and SA. Glycine betaine induced the formation of extracellular globuli containing Ch4. Induced proteins were spatially distributed over the whole plant regardless the site of the inducer application. This revised version was published online in July 2006 with corrections to the Cover Date.     

Simultaneous determination of methamphetamine and its metabolite, amphetamine, in urine using a high performance liquid chromatography column-switching method

We describe here a simple, precise, and highly sensitive method for the simultaneous determination of methamphetamine (MA) and amphetamine (AM) in urine using a high performance liquid chromatography (HPLC) column-switching method. A PK-2A (Shodex) column was used for extraction and deproteinization, and a CAPCELL PAK SCX semi-micro, polymer-coated cation-exchange column was employed for separation. The urine sample was mixed with an equal volume of borate buffer (0.1M, pH 9.4), and then 100 microl of the mixture was injected into the HPLC column. The column was switched for 6 min, and then 10 min later detection was performed at 210 nm. Recovery yields of the MA and AM spiked in the urine were 93.0-100.4% with a coefficient of variation of less than 1%. The calibration curves of MA and AM were in the range of 0.1-10 microg/ml with good linearity (r(2)=0.999), with the limit of qualification being 0.005 microg/ml. This method of using HPLC with column-switching can be used for both qualification and quantification of MA and its metabolite, AM, in urine, especially in forensic cases.     

A novel mixed-mode solid phase extraction for simultaneous determination of melamine and cyanuric acid in food by hydrophilic interaction chromatography coupled to tandem mass chromatography

Utilizing a solid phase extraction column (MCT) containing mixed hydrophilic functional gel and cation exchange sorbent, a sensitive and rapid HPLC–MS/MS method for simultaneously determining the residues of melamine (MEL) and cyanuric acid (CYA) in human foodstuffs was developed. MEL and CYA in egg, pork, liver, kidney and pork, shrimp, sausage casing, honey, soybean milk, soybean powder and dairy product were extracted using acetonitrile/water, defatted with hexane and isolated using MCT solid phase extraction column. The residues were separated upon a hydrophilic interaction liquid chromatography (HILIC) column and analyzed by electrospray ionization under negative–positive switched mode on a triplequadrupole mass spectrometry. The selected reaction monitoring was performed on [M+H]⁺ of m/z 127.9 to provide the transition of 127 > 85 and 127 > 68 (MEL) while the [M−H]⁻ of m/z 127.1 was selected as the precursor ion for CYA resulting in product ions m/z 85 and 42. Isotope labeled internal standard (¹⁵N₃-MEL and ¹³C₃-CYA) and matrix-matched calibration were both used to observe the recovery to be 70.0–129.6% and 70.0–128.9% with RSD of 1.4–23.3% and 1.5–21.7% for MEL and CYA, respectively (n = 6). All the LODs and LOQs of MEL and CYA were less than 39.4 and 99.1 μg kg⁻¹, respectively, in 18 matrices, which were sensitive enough for quantitative analysis. This method has been proven effective in simultaneous determination of melamine and cyanuric acid when inspecting unknown and positive samples.     

Determination of epitestosterone in human urine by off-line immunoaffinity solid-phase extraction coupled with high performance liquid chromatography

Epitestosterone (ET) has been used as a masking agent and prohibited by the World Anti-Doping Agency (WADA) because its administration will decrease the urinary T/ET ratio, a marker of testosterone (T) administration. In this study, an off-line immunoaffinity extraction coupled with high performance liquid chromatography (HPLC) was developed to quantify the endogenous steroid ET in human urine. The immunoaffinity column (IAC) was prepared by immobilizing the anti-ET monoclonal antibodies on CNBr-activated Sepharose 4B, which can remove the contaminations and non-target compounds from matrix to enrich the target analyte ET. The mobile phase was ammonium acetate (10 mM, pH 4.0)/acetonitrile (45/55, v/v) at an isocratic flow of 1.0 mL/min and the UV absorbance detection wavelength was 244 nm for the detection of ET. The IAC showed good reliability and durability since it had been used for more than 100 runs in a year. The limit of quantification (LOQ) was 1 ng/mL. Satisfied repeatability and precision of the day-to-day and within-day were obtained with the RSD values less than 10%. Results of the recovery of the urine samples were ranged from 98% to 102% with repeatability less than 9%, indicating that the method developed can be used for the real urine sample analysis.     

Intrachromosomal homologous recombination in Arabidopsis induced by a maize transposon

In plants, the frequency of spontaneous intrachromosomal homologous recombination is low. Here, we show that a maize transposable element greatly stimulates intrachromosomal homologous recombination between direct repeat sequences in Arabidopsis. Plants were transformed with a construct (GU-Ds-US) containing a Ds (Dissociation) transposable element inserted between two partially deleted GUS reporter gene segments. Homologous recombination between the overlapping GUS fragments generates clonal sectors visible upon staining for GUS activity. Plants containing the GU-Ds-US construct and a source of Ac (Activator) transposase showed an over 1000-fold increase in the incidence of recombination relative to plants containing the same construct but lacking transposase. Transposon-induced recombination was observed in vegetative and floral organs, and several germinally transmitted events were recovered. Transposon-induced recombination appears to be a general phenomenon in plants, and thus may have contributed to genome evolution by inducing deletions between repeated sequences. Received: 28 September 1999 / Accepted: 19 November 1999     

Determination of aesculin in rat plasma by high performance liquid chromatography method and its application to pharmacokinetics studies

A sensitive and reproducible high performance liquid chromatography method with UV detection was described for the determination of aesculin in rat plasma. After deproteinization by methanol using metronidazole as internal standard (I.S.), solutes were evaporated to dryness at 40 degrees C under a gentle stream of nitrogen. The residue was reconstituted in 100 microl of mobile phase and a volume of 20 microl was injected into the HPLC for analysis. Solutes were separated on a Diamonsil C18 column (250 mm x 4.6 mm i.d., 5 microm particle size, Dikma) protected by a ODS guard column (10 mm x 4.0 mm i.d., 5 microm particle size), using acetonitrile-0.1% triethylamine solution (adjusted to pH 3.0 using phosphoric acid) (10:90, v/v) as mobile phase (flow-rate 1.0 ml/min), and wavelength of the UV detector was set at 338 nm. No interference from any endogenous substances was observed during the elution of aesculin and internal standard (I.S., metronidazole). The retention times for I.S and aesculin were 10.4 and 12.4 min, respectively. The limit of quantification was evaluated to be 57.4 ng/ml and the limit of detection was 24.0 ng/ml. The method was used in the study of pharmacokinetics of aesculin after intraperitoneal injection (i.p.) administration in rats.     

Suppression of tumorigenicity and metastasis in murine UV-2237 fibrosarcoma cells by infection with a retroviral vector harboring the interferon-beta gene

 In this study, we endeavored to determine the effectiveness of interferon β (IFNβ) gene therapy against highly metastatic murine UV-2237m fibrosarcoma cells. UV-2237m cells were engineered to produce murine IFNβ constitutively following infection by a retroviral vector harboring the murine IFNβ gene. Parental (UV-2237m-P), control-vector-transduced (UV-2237m-Neo), and IFNβ-transduced (UV-2237m-IFNβ) cells were injected subcutaneously (s.c.) or intravenously (i.v.) into syngeneic mice. Parental and control-transduced cells produced rapidly growing tumors, whereas IFNβ-transduced cells did not. The tumorigenicity of IFNβ-sensitive or -resistant parental cells was significantly suppressed when they were injected s.c. together with IFNβ-transduced cells. The IFNβ-transduced cells did not inhibit growth of parental cells injected s.c. at a distant site. UV-2237m-IFNβ cells produced s.c. tumors in nude, SCID/Beige, and natural killer(NK)-cell-compromised syngeneic mice. The IFNβ-transduced cells were more sensitive to in vitro splenic cell-mediated lysis than were the parental or control-transduced cells. Pretreatment of C3H/HeN mice with the NK-cell-selective antiserum (anti-asialoGM1) partially abrogated the cytotoxic activity of the cells. Cytotoxic activity was not observed in mixed culture of UV-2237m-IFNβ cells and splenic cells from SCID/Beige mice. Significant cytotoxicity against UV-2237m-IFNβ cells was mediated by macrophages activated by either IFNγ, lipopolysaccharide, or a combination of both. Our data led us to conclude that the constitutive expression of IFNβ can suppress tumorigenicity and metastasis of UV-2237m cells, which is due, in part, to activation of host effector cells. Received: 12 November 1997 / Accepted: 30 January 1998     

Identification of two Arabidopsis genes encoding a peroxisomal oxidoreductase-like protein and an acyl-CoA synthetase-like protein that are required for responses to pro-auxins

Indole-3-butyric acid (IBA) and 2,4-dichlorophenoxybutyric acid (2,4-DB) are metabolised by peroxisomal β-oxidation to active auxins that inhibit root growth. We screened Arabidopsis mutants for resistance to IBA and 2,4-DB and identified two new 2,4-DB resistant mutants. The mutant genes encode a putative oxidoreductase (SDRa) and a putative acyl-activating enzyme (AAE18). Both proteins are localised to peroxisomes. SDRa is coexpressed with core β-oxidation genes, but germination, seedling growth and the fatty acid profile of sdra seedlings are indistinguishable from wild type. The sdra mutant is also resistant to IBA, but aae18 is not. AAE18 is the first example of a gene required for response to 2,4-DB but not IBA. The closest relative of AAE18 is AAE17. AAE17 is predicted to be peroxisomal, but an aae17 aae18 double mutant responded similarly to aae18 for all assays. We propose that AAE18 is capable of activating 2,4-DB but IBA activating enzymes remain to be discovered. We present an updated model for peroxisomal pro-auxin metabolism in Arabidopsis that includes SDRa and AAE18. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A rapeseed FAE1 gene is linked to the E1 locus associated with variation in the content of erucic acid

 The synthesis of very long chain fatty acids occurs in the cytoplasm via an elongase complex. A key component of this complex is the β-ketoacyl-CoA synthase, a condensing enzyme which in Arabidopsis is encoded by the FAE1 gene. Two sequences homologous to the FAE1 gene were isolated from a Brassica napus immature embryo cDNA library. The two clones, CE7 and CE8, contain inserts of 1647 bp and 1654 bp, respectively. The CE7 gene encodes a protein of 506 amino acids and the CE8 clone, a protein of 505 amino acids, each having an approximate molecular mass of 56 kDa. The sequences of the two cDNA clones are highly homologous yet distinct, sharing 97% nucleotide identity and 98% identity at the amino acid level. Southern hybridisation showed the rapeseed β-ketoacyl-CoA synthase to be encoded by a small multigene family. Northern hybridisation showed the expression of the rapeseed FAE1 gene(s) to be restricted to the immature embryo. One of the FAE1 genes is tightly linked to the E1 locus, one of two loci controlling erucic acid content in rapeseed. The identity of the second locus, E2, is discussed. Received: 4 April 1997 / Accepted: 30 July 1997     

Speciation of trace elements in proteins in human and bovine serum by size exclusion chromatography and inductively coupled plasma-mass spectrometry with a magnetic sector mass spectrometer

 Proteins are separated by size exclusion chromatography while atomic ions from the inorganic elements are detected on-line by inductively coupled plasma-mass spectrometry. A double focusing mass analyzer provides very high sensitivity, low background, and sufficient spectral resolution to separate the atomic ions of interest from most polyatomic ions at the same nominal m/z value. The chromatograms show the distribution of the elements of interest between protein-bound and free fractions and provide the approximate molecular weights of those protein fractions that contain the elements monitored. The distribution of various elements, including V, Mo, Fe, Co, Mn, and lanthanides, in human or bovine serum samples are shown. Alkali metals and Tl are present primarily as free metal ions and are not bound to proteins. Inorganic elements spiked into the serum samples can be followed into various proteins. EDTA does not remove Fe, Pb, Sn, or Th from the proteins but does extract Mn from some proteins. Procedures for determining the effects of breaking disulfide linkages on the metal binding characteristics of proteins are also described. Received: 22 March 1999 / Accepted: 16 June 1999