High Resolution Melting Analysis: A Rapid Screening and Typing Tool for Common β-Thalassemia Mutation in Chinese Population

β-thalassemia is a common inherited disorder worldwide including southern China, and at least 45 distinct β-thalassemia mutations have been identified in China. High-resolution melting (HRM) assay was recently introduced as a rapid, inexpensive and effective method for genotyping. However, there was no systemic study on the diagnostic capability of HRM to identify β-thalassemia. Here, we used an improved HRM method to screen and type 12 common β-thalassemia mutations in Chinese, and the rapidity and reliability of this method was investigated. The whole PCR and HRM procedure could be completed in 40 min. The heterozygous mutations and 4 kinds of homozygous mutations could be readily differentiated from the melting curve except c.-78A>G heterozygote and c.-79A>G heterozygote. The diagnostic reliability of this HRM assay was evaluated on 756 pre-typed genomic DNA samples and 50 cases of blood spots on filter paper, which were collected from seven high prevalent provinces in southern China. If c.-78A>G heterozygote and c.-79A>G heterozygote were classified into the same group (c.-78&79 A>G heterozygote), the HRM method was in complete concordance with the reference method (reverse dot blot/DNA-sequencing). In a conclusion, the HRM method appears to be an accurate and sensitive method for the rapid screening and identification of β-thalassemia mutations. In the future, we suggest this technology to be used in neonatal blood spot screening program. It could enlarge the coverage of β-thalassemia screening program in China. At the same time, its value should be confirmed in prospectively clinical and epidemiological studies.     

Regulation of purine biosynthetic genes expression in Salmonella typhimurium Ⅳ Oc mutation site of purG and its function analysis

Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded bypurG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu- tamine and ATP for thede novo purine nucleotide biosynthesis.purG gene is negatively regulated by a repressor-operator system. The O⁺ purG and O^c purG were cloned respectivelyin vivo. Restriction enzymes analysis of preliminary clones pLBG-1 (O⁺) and pLBG-2 (O^c) were carried out. The hybrid plasmids pLB1933 (O⁺) and pLB1927 (O^c) containing 5′ control region ofpurG were constructed and the DNA sequences were determined respectively, DNA sequences data showed that O^c mutation ofpurG occurred at the 3rd position of 16 bp PUR box in the 5′ control region (G→A). Gel retardation experiment indicated that the repressor bound well with O⁺ PUR box, but not with O^c PUR box. The result strongly supported the idea that PUR box is the binding region of repressor protein and the 3rd position base G of PUR box is essential for the binding function with repressor protein. Project supported by the National Natural Science Foundation of China.     

Molecular cloning and expression analysis of three genes encoding starch synthase II in rice

Three starch synthase (SS) genes, OsSSII-1, OsSSII-2 and OsSSII-3, were identified in rice (Oryza sativa L.) and localized to chromosomes 10, 2 and 6, respectively. The three OsSSII full-length cDNAs were cloned, and the predicted amino acid sequences were found to share 52–73% similarity with other members of the plant SSII family. The SS activity of each OsSSII was confirmed by expression and enzyme activity assay in Escherichia coli. Expression profile analysis revealed that OsSSII-1 was expressed in endosperms, leaves and roots; OsSSII-2 was mainly expressed in leaves, while OsSSII-3 was mainly expressed in endosperms. Similar to the OsSSI proteins, the OsSSII-2 and OsSSII-3 proteins were found in the soluble as well as the starch-granule-bound fractions in rice. The roles of the OsSSII proteins in starch biosynthesis in rice and the evolutionary relationships of the genes encoding monocotyledonous and dicotyledonous class-II SS enzymes are discussed.Abbreviations CDS Coding domain sequence - EST Expressed sequence tag - GB Granule-bound - Glc Glucose - SS Starch synthase     

Simultaneous determination of a novel antifibrotic agent and three metabolites in human urine by LC–MS/MS

A robust and validated high performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for simultaneous determination of F351 (5-methyl-1-(4-hydroxylphenyl)-2-(1H)-pyridone) and three major metabolites in human urine sample. This assay method has also been validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect and recovery. Chromatography was carried out on an XTerra RP 18 column and mass spectrometric analysis was performed using an API 4000 mass spectrometer coupled with electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 202 → 109, 232 → 93, 282 → 202 and 378 → 202 were used to quantify F351 and three metabolites, respectively. Retention times for F351 and three metabolites were 2.54, 1.38, 1.53 and 1.34 min, respectively. The assay was validated from 20 to 4000 ng/mL for F351 and M1, from 80 to16,000 ng/mL for M2 and M3. Intra- and inter-day precision for all analytes was <6.3%, method accuracy was between −11.2 and 0.3%. This assay was used to support a clinical study where multiple oral doses were administered to healthy subjects to investigate the pharmacokinetics, safety, and tolerability of F351.     

Simultaneous determination of m-nisoldipine and its three metabolites in rat plasma by liquid chromatography–mass spectrometry

A simple, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of m-nisoldipine and its three metabolites in rat plasma has been developed using nitrendipine as an internal standard (IS). Following liquid–liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C₁₈ column and analyzed by MS in the multiple reaction monitoring (MRM) mode. To avoid contamination by residual sample in the injection syringe, a special injection protocol was developed. We found that m-nisoldipine, metabolite M1 and IS could be ionized under positive or negative electrospray ionization conditions, whereas metabolite M and M2 could only be ionized in the positive mode. The mass spectrometry fragmentation pathways for these analytes are analyzed and discussed herein. The total analysis time required less than 5 min per sample. We employed this method successfully to study the metabolism of m-nisoldipine when it was orally administered to rats at a dose of 9 mg/kg. Three metabolites of m-nisoldipine and an unknown compound of molecular weight 386 were found for the first time in rat plasma. The concentration of the potentially active metabolite was approximately equal to its parent compound concentration.     

Regulation of purine biosynthetic genes expression in Salmonella typhimurium Ⅳ O~c mutation site of purG and its function analysis

Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded by purG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu-tamine and ATP for the de novo purine nucleotide biosynthesis. purG gene is negatively regulated by a repressor-oper-ator system. The O purG and OC purG were cloned respectively in vivo. Restriction enzymes analysis of preliminary clones pLBG-1 (O ) and pLBG-2 (OC) were carried out. The hybrid plasmids pLB1933 (O ) and pLB1927 (OC) containing 5 control region of purG were constructed and the DNA sequences were determined respectively. DNA se-quences data showed that Oc mutation of purG occurred at the 3rd position of 16 bp PUR box in the 5' control region ( G→A). Gel retardation experiment indicated that the repressor bound well with O PUR box, but not with Oc PUR box. The result strongly supported the idea that PUR box is the binding region of represser protein and the 3rd posi-tion base G of PUR bo     

Simultaneous detection of residues of β-adrenergic receptor blockers and sedatives in animal tissues by high-performance liquid chromatography/tandem mass spectrometry

A method using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) was developed to screen and confirm residues of multi-class veterinary drugs in animal tissues (porcine kidney, liver, muscle; bovine muscle). Thirty target drugs (19 β-blockers, 11 sedatives) were determined simultaneously in a single run. Homogenized tissue samples were extracted with acetonitrile and purified using a NH₂ solid-phase extraction cartridge. An Acquity UPLC? BEH C18 column was used to separate the analytes, followed by tandem mass spectrometry using an electrospray ionization source in positive mode. Recovery studies were done at three fortification levels. Overall average recoveries in pig muscle, kidney, and liver fortified at three levels from 76.4% to 118.6% based on matrix-fortified calibration with coefficients of variation from 2.2% to 19.9% (n = 6). The limit of quantification of these compounds in different matrices was 0.5–2.0 μg/kg. This method was successfully applied in screening and confirming target drugs in >200 samples.     

Restoration by the rac locus of recombinant forming ability in recB? and recC? merozygotes of Escherichia coli K-12

Summary The recombinant forming ability of recB ^– or recC ^– strains of E. coli K12 is almost totally recovered in merozygotes which are heterozygous for a genetic locus denoted rac which is located five minutes clockwise from trp on the genetic map. This transient recovery phenomenon only occurs when the donor strain is rac ⁺ (wild type) and the recipient strain is rac ^-. The recombinants derived from such crosses all have the normal phenotype characteristic of recB ^– (or recC ^–) strains, and they are almost always rac ^-. The results imply that the rac ⁺ locus (or loci) is zygotically expressed and excised from the chromosome in a manner which is analogous to the zygotic induction of a prophage.     

Simultaneous determination of histamine and Nτ-methylhistamine in human plasma and urine by gas chromatography-mass spectrometry

A new and sensitive method is described for the determination of histamine and N^τ-methylhistamine in human plasma and urine by gas chromatography-mass spectrometry. ¹⁵N₂-Labeled histamine and N^τ-[methyl-d₃]methylhistamine were used as internal standards. Histamine and N^τ-methylhistamine were converted to the derivatives N^α-heptafluorobutyryl-N^τ-ethoxycarbonylhistamine and N^α-heptafluorobutyryl-N^τ-methylhistamine, respectively. After these derivatives had been purified on a small column packed with CPG-10, the molecular ions were monitored during selected ion monitoring. Linear standard curves were obtained in the range of 0.5–10 ng/ml for both compounds. The reliability of the histamine analysis was demonstrated by using two different ion pairs, while a comparison with results from two different derivatizations on the same urine sample also established the specificity of the N^τ-methylhistamine analysis. An increase of 1 ng of histamine in the plasma could be precisely determined by the present method. The histamine content of plasma from five normal subjects was determined as 0.83 ÷ 0.37 (S.D.) ng/ml and the N^τ-methylhistamine content in most subjects was below the limits of this measurement. High excretion of histamine was noted in the urine collected in the early morning from a patient with nephritis.     

Genome change in wheat observed through the structure and expression of α/β-gliadin genes

To better understand genome structure and the expression of α/β-gliadin multigenes in hexaploid wheat, bacterial artificial chromosome (BAC) clones containing α/β-gliadin genes from the three loci, Gli-A2, Gli-B2, and Gli-D2, were screened. Based on their restriction fragment patterns, we selected five BAC clones, namely, two clones for Gli-A2, two clones for Gli-B2, and one clone for Gli-D2, to fully sequence. Approximately 200 kb was sequenced for each locus. In total, twelve α/β-gliadin intact genes and four pseudogenes were found, and retrotransposons or other transposons existed in each BAC clone. Dot-plot analysis revealed the pattern of genome segmental duplication within each BAC. We calculated time since duplication of each set of α/β-gliadin genes and insertion of retrotransposons. Duplication of all adjacent genes within the same BAC clone took place before or after allotetrapolyploidization, but duplication of certain genes occurred before diploid differentiation of wheat species. Retrotransposons were also inserted before and after the segmental duplication events. Furthermore, translocation of α/β-gliadin genes from chromosomes 1 to 6 apparently occurred before the diversification of various wheat genomes. Duplication of genome segments containing α/β-gliadin genes and retrotransposons were brought about through unequal crossing-over or saltatory replication and α/β-gliadin genes per se were duplicated without any recombination events. Out of twelve intact α/β-gliadin genes detected from their sequences, nine were expressed, although their patterns of expression were distinct. Since they have similar cis-elements and promoter structures, the mechanisms underlying their distinct gene expression and possible applications are discussed.     

Activation of phosphates to substitution(II). A comparison of catalyses initiated directly by VO2+ and by oxidation of VO2+ by MnO4−

Vanadium(V)-induced hydrolyses of triphosphates in aqueous solutions were initiated in two ways: (1) oxidizing vanadium(IV)-polyphosphate complexes to produce metastable vanadium(V) complexes; (2) forming VO₂⁺-polyphosphate complexes by acidification of solutions of VO₄^3? and polyphosphate to yield equilibrium mixtures of V(V), polyphosphate, and their complexes. Hydrolysis rates for the complexes formed at 40°C ? T ? 25°C follow the order V₂PPP_i = 2(VPPP_i) ≌ (VO₂) ATP ? V(PPP_i)₂ ≌ PPP_i. The hydrolysis of (V(V))₂(PPP_i) was not very temperature sensitive; the activation enthalpy appears small and the activation entropy large and negative. Mechanistic studies reveal that requirements for the activated state in metal-ion-catalyzed hydrolysis of polyphosphates include monodentate polyphosphate ligated cis to H₂O or OH^? in the coordination sphere of the metal ion:

     

Electrically silent cotransport of Na+, K+ and Cl− in ehrlich cells

A cotransport system for Na⁺, K⁺ and Cl^? in Ehrlich cells is described. It is insensitive towards ouabain but specifically inhibited by furosemide and other ‘high ceiling’ diuretics at concentrations which do not affect other pathways of the ions concerned. As the furosemide-sensitive fluxes of these ions are not affected by changes in membrane potential, and as their complete inhibition by furosemide does not appreciably alter the membrane potential, they appear to be electrically silent. Application of the pulse-response methods in terms of irreversible thermodynamics reveals tight coupling between the furosemide-sensitive flows of Na⁺, K⁺ and Cl^? ($\text{q}$ close to unity for all three combinations) at a stoichiometry of 1 : 1 : 2. The site for each of the ions appears to be rather specific: K⁺ can be replaced by Rb⁺ but not by other cations tested whereas Cl^? can be poorly replaced by Br^? but not by NO₃^?, in contradistinction to the Cl^?-OH^? exchange system. The cotransport system appears to function in cell volume regulation as it tends to make the cell swell, thus counteracting the shrinking effect of the ouabain-sensitive (Na⁺, K⁺) pump.The experiments presented could not clarify whether the cotransport process is a primary or secondary active one; while incongruence between transport and conjugated driving force seems to indicate primary active transport, it is very unlikely that hydrolysis of ATP supplies energy for the transport process, since there is no stimulation of ATP turnover observable under operation of the cotransport system.     

Simultaneous characterization of sequence polymorphisms,glycosylation and phosphorylation of fibrinogen in a direct analysis by LC–MS

Fibrinogen is an abundant plasma glycoprotein involved in pathologically important processes like blood clotting, hemostasis and angiogenesis. Sequence polymorphisms and posttranslational modification (PTM) status of fibrinogen are important factors of cardiovascular disease. We aim for the simultaneous analysis of fibrinogen subunits for sequence polymorphisms (SNPs), phosphorylation and glycosylation by top-down mass spectrometry. Fibrinogen was isolated from human plasma of twelve individuals and subunits of fibrinogen were separated by RP-HPLC and subsequently analyzed by high resolution ESI mass spectrometry. Two coding single nucleotide polymorphisms on the Aα- and Bβ-subunit could be identified on the basis of their mass shifts: Three individuals are heterozygous and two are homozygous for Thr312Ala on the Aα-subunit, three individuals are heterozygous for Arg448Lys on the Bβ-subunit. For the Aα-subunit we find mono- and diphosphorylation amounting to about 55% to 71% and O-glycosylation (likely sialyl-T-antigen) from 10% to 17%. N-glycosylation is present with one or two sialic acids in a ratio of about 3:2 and 3:1 for the Bβ and the γ-subunit, respectively. Both SNPs and the PTMs are associated with fibrinogen levels, clotting behavior and thus the risk for cardiovascular diseases. The homozygosity of the SNP at position 312 in the alpha chain for example nearly doubles the risk for ischemic stroke. Isolation and analysis of fibrinogen can be achieved in a few hours from only one drop of blood plasma, and thus the method presented here should assist in a quick assessment and prevention of stroke and infarction.     

Expression of the plasmid pKM101-determined DNA repair system in recA? and lex? strains of Escherichia coli

Summary pKM101, a plasmid R factor of the N compatibility group increases methylmethane sulfonate mutagenesis and diminishes UV-killing in recA ⁺ lex ⁺ and recA ⁺ lex ^– strains, but not in recA ^– lex ⁺ strains. The induction of a reclex dependent colicin is not present in lex ^– strains carrying the pKM101 factor. These facts indicate that pKM101 acts through an error-prone DNA repair system, which is recA ⁺ dependent, but not lex ⁺ dependent.This paper is published on the occasion of Dr. C. Callerio's seventy-fifth birthday     

Simultaneous determination of type A-& B-trichothecenes and zearalenone in cereals by High Performance Liquid Chromatography — Tandem Mass Spectrometry

A rapid quantitative method for the simultaneous determination of the majorFusarium mycotoxins nivalenol, deoxynivalenol, fusarenon-X, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, diacetoxyscirpenol, HT-2 toxin, T-2 toxin and zearalenone in maize and wheat was developed. Raw extracts (acetonitrile/water 84/16) are cleaned-up with MycoSep^® columns., Chromatographic separation and end determination is carried out by HPLC-APCI-MS/MS.HPLC run times of 10 minutes considerably increases sample throughput and make this method suitable for routine analysis. The use of a triple quadrupole mass spectrometer allows the selective detection of the mycotoxins and their quantification in the low μg/kg-range.     

Quantitative analysis of methylguanidine and guanidine in physiologic fluids by high-performance liquid chromatography—fluorescence detection method

A rapid and sensitive high-performance liquid chromatographic method has been developed for the quantitative analysis of methylguanidine and guanidine in physiological fluids. These guanidino compounds are separated on a 6 × 0.23 cm cation-exchange column with 0.5 M sodium hydroxide solution. The guanidino compounds are detected with a fluorometer, which monitors the fluorescent guanidine derivatives produced by the reaction of the eluted constituents with 9,10-phenanthrenequinone. Sensitivity to sub-nanomole levels of methylguanidine and guanidine is demonstrated. The method was successfully applied to physiological fluids such as serum and cerebrospinal fluid from uremic patients.