Selfcompatibility in microspore-derived doubled-haploid rye lines and single grain selection for alkylresorcinol content

Summary From a total of 138 green androgenetic rye lines, 25 were fertilized and examined in field tests: 7 were heterozygous and 18 were homozygous. Of the homozygotes, 4 turned out to be selfincompatible, while 14 set seed after selfing. Four characters were analyzed in detail: 100 kernel weight, plant height, ear length, and alkylresorcinol content. Here we present the first approach in prescreening selfcompatible androgenetic doubled-haploid rye plants with the single grain procedure. The usefulness of this method was confirmed by quantitative resorcinol determination in the following generation. Furthermore, it was demonstrated that all the homologues of the alkylresorcinol were equally reduced. For all characters the means of the different anther derived lines exceeded the means of the controls in both directions, to the positive as well as to the negative side. The incorporation of such a haploid breeding step into breeding programs is discussed.Dedicated to Professor Dr. G. Melchers, on the occasion of his 75th birthday, who initiated this work by forming the project-groups Haploide in der Pflanzenzüchtung     

Identification of albumin 0.19 in wheat and other cereal proteins

It was found that albumin 0.19, being an inhibitor of alpha-amylases from human and insect saliva, is not specific for T. aestivum and is also revealed immunochemically by means of antiserum in the proteins of T. durum. Using rocket immunoelectrophoresis, it was shown that the albumin component specific for T. aestivum is not identical to albumin 0.19. The levels of albumin 0.19 in the proteins of wheat, aegilops. secale, agropyron and barley grains were studied.     

Mining biomarkers in human sera using proteomic tools

One of the major difficulties in mining low abundance biomarkers from serum or plasma is due to the fact that a small number of proteins such as albumin, alpha2-macroglobulin, transferrin, and immunoglobulins, may represent as much as 80% of the total serum protein. The large quantity of these proteins makes it difficult to identify low abundance proteins in serum using traditional 2-dimensional electrophoresis. We recently used a combination of multidimensional liquid chromatography and gel electrophoresis coupled to matrix-assisted laser desorption/ionization-quadrupole-time of flight and Ion Trap liquid chromatography-tandem mass spectrometry to identify protein markers in sera of Alzheimer's disease (AD), insulin resistance/type-2 diabetes (IR/D2), and congestive heart failure (CHF) patients. We identified 8 proteins that exhibit higher levels in control sera and 36 proteins that exhibit higher levels in disease sera. For example, haptoglobin and hemoglobin are elevated in sera of AD, IR/D2, and CHF patients. The levels of several other proteins including fibrinogen and its fragments, alpha 2-macroglobulin, transthyretin, pro-platelet basic protein, protease inhibitors clade A and C, as well as proteins involved in the classical complement pathway such as complement C3, C4, and C1 inhibitor, were found to differ between IR/D2 and control sera. The sera levels of proteins, such as the 10 kDa subunit of vitronectin, alpha 1-acid glycoprotein, apolipoprotein B100, fragment of factor H, and histidine-rich glycoprotein were observed to be different between AD and controls. The differences observed in these biomarker candidates were confirmed by Western blot and the enzyme-linked immunosorbent assay. The biological meaning of the proteomic changes in the disease states and the potential use of these changes as diagnostic tools or for therapeutic intervention will be discussed.     

Identification of putative serum glycoprotein biomarkers for human lung adenocarcinoma by multilectin affinity chromatography and LC-MS/MS

Glycoproteins in human serum play fundamental roles in many biological processes, and also have clinical value as biomarkers for disease progression and treatment. In this study, we isolated glycoproteins from the sera of three healthy individuals and three lung adenocarcinoma patients using multilectin affinity chromatography. The recovered glycoproteins were subjected to treatment with peptide-N-glycosidase F (PNGase F) and in-gel digestion by trypsin. Tryptic peptides were analyzed by nano-LC coupled to ESI-MS/MS and the MS/MS spectra were processed by Bioworks 3.2 and an in-house bioinformatics tool, ProtAn. Approximately 90% of the proteins identified contained more than one potential glycosylation site. Comparison of the serum glycoproteome of healthy and adenocarcinoma individuals revealed 38 cancer-selective proteins. Among them, 60% have previously been reported as low abundance proteins in human sera. We identified several cancer-selective proteins that have been previously characterized as potential indicators of lung cancer in serum or plasma, including haptoglobin (HP), inter-alpha-trypsin inhibitor heavy chain 4 (ITI-H4), complement C3 precursor, and leucine-rich alpha-2-glycoprotein. In addition, plasma kallikrein (KLKB1) and inter-alpha-trypsin inhibitor heavy chain 3 (ITI-H3) were identified as being potentially elevated in the lung cancer group, and were validated by Western blot analysis. Furthermore, approximately 18 kDa plasma kallirein protein fragment was detected at high levels in 25 out of 28 adenocarcinoma patients, while one of the eight normal individuals showed moderate positive. The results suggest that KLKB1 represents a potential candidate serum biomarker of lung cancer.     

Identification of a repeat sequence of rye DNA in wheat and related species

Polymerase chain reaction (PCR) and enhanced chemiluminescence (ECL) were used to determine the distribution of the rye-specific sequence contained in the pSc119.1 probe among wheat and related species. A specific pair of primers targeting this rye-specific sequence was used. A 745-bp fragment, the predicted size of pSc119.1, was present inSecale cereale, Triticum aestivum, XTriticosecale, Hordeum vulgare, H. bogdanii, andH. parodii. PCR results were verified by hybridizing the rye-specific probe pSc119.1 to dotblots of DNA from the different species used. Strong hybridization signals detected by ECL were consistent with the PCR results. The results demonstrate the effectiveness of PCR and dot-blot ECL in screening plants for defined DNA sequences, and indicate that pSc119.1 has counterparts with strong homology inT. aestivum, H. vulgare, H. bogdanii, andH. parodii.     

Identification of selenosugars and other low-molecular weight selenium metabolites in high-selenium cereal crops

Several novel selenium containing compounds were characterized in staple crops (wheat, rice and maize) grown on soils naturally rich in selenium. A dedicated method based on the coupling of liquid chromatography with multiplexed detection (ICP-MS, ESI-Orbitrap MS(/MS)) was developed for the speciation of low-molecular weight (<5 kDa) selenium metabolites. Nine species present in different proportions as a function of the crop type were identified by cation-exchange HPLC-ESI-Orbitrap MS on the basis of the accurate molecular mass and MS/MS spectra. The natural origin of these species was then validated by varying extraction conditions and by using hydrophilic interaction LC (HILIC)-ESI-Orbitrap MS(/MS). Among the identified compounds, Se-containing monosaccharides (hexose moiety, m/z 317 and m/z 358) or Se-containing disaccharides (hexose-pentose moiety, m/z 407 and m/z 408) were the first selenosugars reported in edible plants. It is also the first report of the presence of 2,3-dihydroxypropionyl-selenolanthionine (m/z 345) in rice. Because these crops can be an important source of selenium in animal and human nutrition, the understanding of the origin and the fate of these species during metabolic processes will be of great interest.     

Identification of biomarkers for early diagnosis of Parkinson's disease by multi-omics joint analysis

ObjectiveThe purpose of this study is to identify the biomarkers for early diagnosis of Parkinson's disease (PD) by multi-omics joint analysis, so as to identify the biomarkers for early diagnosis of PD, and to help clinicians make early diagnosis and treatment.MethodsIn this study, mice are taken as the study subjects. The model of PD mice is established, and then lymphocyte, striatum, substantia nigra protein and proteolysis are extracted. After that, the experiments of protein imprinting and 4¹⁸O labeling are carried out. Mass Spectrometry (MS) analysis technology is mainly used to study proteomics and to analyze the quantitative and qualitative situation of differential proteins in striatum, substantia nigra protein and lymphocyte. By this method, biomarkers for early diagnosis of PD are analyzed and identified.ResultsThe biomarkers of Parkinson's early onset are related to the same quantitative differential expression of lymphocyte, striatum, substantia nigra protein, lymphocyte and substantia nigra.ConclusionThis experimental method can analyze and identify the biomarkers of early diagnosis of PD, help to explore the pathophysiology and pathogenesis of PD, effectively help clinicians make timely diagnosis in advance, and improve the prevention and treatment effect of the disease.     

Dissecting the human plasma proteome and inflammatory response biomarkers

A central focus of clinical proteomics is to search for biomarkers in plasma for diagnostic and therapeutic use. We studied a set of plasma proteins accessed from the Healthy Human Individual's Integrated Plasma Proteome (HIP²) database, a larger set of curated human proteins, and a subset of inflammatory proteins, for overlap with sets of known protein biomarkers, drug targets, and secreted proteins. Most inflammatory proteins were found to occur in plasma, and over three times the level of biomarkers were found in inflammatory plasma proteins and their interacting protein neighbors compared to the sets of plasma and curated human proteins. Percentage overlaps with Gene Ontology terms were similar between the curated human set and plasma protein set, yet the set of inflammatory plasma proteins had a distinct ontology‐based profile. Most of the major hub proteins within protein‐protein interaction networks of tissue‐specific sets of inflammatory proteins were found to occur in disease pathways. The present study presents a systematic approach for profiling a plasma subproteome's relationship to both its potential range of clinical application and its overlap with complex disease.     

Identification of the 1RS rye chromosomal segment in wheat by RAPD analysis

The introgression of rye DNA into the wheat genome was studied using random decamer and specific primers with the polymerase chain reaction (PCR). DNA from paired near-isolines in Chisholm and Arkan backgrounds differing with respect to the presence of a 1 RS.1 BL translocation was amplified with 120 arbitrary sequence primers. Two of the primers (OPR 19 and OPJ07) amplified rye-specific DNA fragments. The OPR19 primer amplified a 1.35-kb fragment that appeared to be specific to the 1 RS.1 BL translocation, based on its presence only in lines carrying the 1 RS. 1 BL translocation. A fragment of the same size was also amplified in 1 RS.1 AL translocation lines. This 1 RS. 1 BL marker locus was designated Ximc 1. The other primer, OPJ07, amplified a 1.2-kb DNA sequence, that was designated Ximc 2, specific to the wheat-rye translocation in various wheat backgrounds. The sequences of the two marker loci were found to be different from each other. The Ximc 1 locus was a low-copy sequence which was also present in Balboa rye genomic DNA. Through the use of specific primers, the presence of the rye-specific marker was confirmed in hexaploid as well as in tetraploid wheat backgrounds. The use of RAPDs for the study of smaller alien introgressions into wheat is discussed.     

Identification and sequence analysis of grain softness protein in selected wheat, rye and triticale

Grain softness protein (GSP) is an important protein for overcoming milling and grain defenses in the innate immunity systems of cereals. The objective of this study was to evaluate and understand GSP sequences in selected wheat, rye and triticale. Using sequences for this gene from a sequence database, we performed clustering analysis to compare the sequences obtained from 3 germplasms with other studied sequences for GSP. The maximum difference between the Hirmand GSP genotype in wheat and the database sequences was 23% in EF109396 and EF109399. Most amino acid variation between the GSP sequences involved the same amino acids. The Nikita rye GSP gene showed 64% identity with DQ269918 and AY667063. The isoelectric point in the GSP of wheat and Lasko triticale was significantly higher than that of rye GSP. In addition, parameters such as optical density, grand average of hydrophobicity, percentage of hydrophobicity and hydrophilic amino acids, and number of alpha helices and beta sheets in GSP were similar in wheat and triticale but not in wheat and rye.     

Identification of fendiline metabolites in human urine

After oral application of 14C labelled fendiline, 13 metabolites of this drug could be identified in human urine. Only traces of parent fendiline were excreted in the urine. The main pathway of metabolism is hydroxylation of phenyl groups with subsequent glucuronidation and sulphation. On the other hand, oxidative dealkylation occurs with the amino group remaining at the 3,3-diphenylpropyl moiety and p-hydroxyacetophenone being formed almost entirely from the 1-phenylethyl group.     

Chiasma distribution in rye chromosomes of diploid rye and in wheat/rye addition lines in relation to C-heterochromatin

Summary In five genetically different inbred lines of rye and in the seven Chinese Spring/Imperial wheatrye addition lines, chiasma distribution in rye chromosomes was studied with respect to the amount and position of constitutive heterochromatin (Giemsa C-bands). In all inbred lines, rye chromosomes with one primary terminal band were more frequently found as univalents than those with primary bands on both telomeres. These chromosomes were most probably 5R and/or 6R. In the addition lines a highly significant reduction in the number of arms bound by chiasmata was found for rye chromosomes 5R and 6R. Because of the similar chiasma distribution in the inbred lines and in the rye chromosomes of the addition lines, no effect of the wheat genome on the number of chiasmata in the rye chromosomes can be ascertained. However, a relationship between chiasma frequency and chromosome arm length seems to exist, since under reduced chiasma conditions the two shortest arms of the rye complement, those of chromosomes 5R and 6R, frequently fail to form a chiasma. No effect of the large blocks of constitutive heterochromatin in the telomeres of the rye chromosomes on the position of chiasmata within a bivalent could be established.This study was financially supported by the Deutsche Forschungsgemeinschaft     

Cytogenetic investigations in rye,wheat and triticale

Summary Chromosomes of tetra- and hexaploid wheat have been individually characterized by Giemsa and/or Leishman C-banding techniques. Appropriate methodological modifications resulted in almost identical staining of chromosomes of tetraploid wheat with Giemsa and Leishman solutions. Additionally comparison of Giemsa banded chromosomes of the A- and B-genome of Triticum turgidum 34 and Triticum aestivum cv Jubilar reveals similar or corresponding patterns in all homologous chromosomes with the exception of chromosome 7B. Apart from this intervarietal variation in certain homologous chromosomes of both wheat cultivars, intravarietal polymorphism is verified.     

人类蛋白组学草图的肺癌分子标记物初探

传统的肺癌分子标记物探索通常基于基因组或者转录组研究,而基于蛋白质水平的肺癌分子标记物探索通常局限在低通量水平。质谱技术已经开始产生高通量的全局正常及癌症蛋白组。我们采用开源统计软件R对人类蛋白组学草图数据及已发表的肺癌蛋白质组学数据进行二次分析,筛选出91个潜在的候选肺癌分子标记物。基因注解分析显示候选肺癌基因富集了和代谢、TP53通路以及MicroRNA调控等相关的基因。最后,利用Human Protein Atlas数据库及Pubmed对前20候选标记物进行验证,结果显示大部分候选肺癌基因大多能够得到验证。可见数据挖掘在即将到来的质谱推动的组学大数据时代将发挥重要作用。     

Identification of potential serum biomarkers of inflammation and lipid modulation that are altered by fish oil supplementation in healthy volunteers

Long chain n-3 polyunsaturated fatty acids (n-3 LCPUFA) lower risk of coronary heart disease (CHD), but mechanisms are not well understood. We used proteomics to identify human serum proteins that are altered by n-3 LCPUFA. Such proteins could identify pathways whereby they affect CHD. Eighty-one healthy volunteers entered a double blind randomised trial to receive 3.5 g of fish oil or 3.5 g of high oleic sunflower oil daily. Serum was collected before and after 6 wk of intervention. Serum was analysed by proteomics using 2-DE. Proteins that were differentially regulated were identified by MS. We also analysed serum apolipoprotein A1 (apo A1), high-density lipoprotein (HDL) particle size and haptoglobin. Serum levels of apo A1, apo L1, zinc-alpha-2-glycoprotein, haptoglobin precursor, alpha-1-antitrypsin precursor, antithrombin III-like protein, serum amyloid P component and haemopexin were significantly downregulated (all p<0.05) by fish oil compared with high oleic sunflower oil supplementation. Fish oil supplementation caused a significant shift towards the larger, more cholesterol-rich HDL(2) particle. The alterations in serum proteins and HDL size imply that fish oil activates anti-inflammatory and lipid modulating mechanisms believed to impede the early onset of CHD. These proteins are potential diagnostic biomarkers to assess the mechanisms whereby fish oil protects against CHD in humans.     

Identification and chromosomal organization of two rye genome-specific RAPD products useful as introgression markers in wheat.

Two rye genome-specific random amplified polymorphic DNA (RAPD) markers were identified for detection of rye introgression in wheat. Both markers were amplified in all of the tested materials that contained rye chromatin such as rye, hexaploid triticale, wheat-rye addition lines, and wheat varieties with 1BL.1RS translocation. Two cloned markers, designated pSc10C and pSc20H, were 1012 bp and 1494 bp, respectively. Sequence analysis showed that both pSc10C and pSc20H fragments were related to retrotransposons, ubiquitously distributed in plant genomes. Using fluorescence in situ hybridization (FISH), probe pSc10C was shown to hybridize predominantly to the pericentromeric regions of all rye chromosomes, whereas probe pSc20H was dispersed throughout the rye genome except at telomeric regions and nucleolar organizing regions. The FISH patterns showed that the two markers should be useful to select or track all wheat-rye translocation lines derived from the whole arms of rye chromosomes, as well as to characterize the positions of the translocation breakpoints generated in the proximal and distal regions of rye arms.     

Induction of small-segment-translocation between wheat and rye chromosomes

A new approach to produce wheat-rye translocation, based on the genetic instability caused by monosomic addition of rye chromosome in wheat, is described. 1 283 plants from the selfed progenies of monosomic addition lines with single chromosome of inbred rye line R12 and complete chromosome complement of wheat cultivar Mianyang 11 were cytologically analyzed on a plant-by-plant basis by the improved C-banding technique. 63 of the plants, with 2n = 42, were found containing wheat-rye translocation or substitution, with a frequency of 4. 91% . Compared with the wheat parent, other 32 plants with 2n = 42 exhibited obvious phenotypic variation, but their com-ponent of rye chromosome could not be detected using the C-banding technique. In situ hybridization with a biotin-la-beled DNA probe was used to detect rye chromatin and to determine the insertion sites of rye segments in the wheat chromosomes. In 20 out of the 32 variant wheat plants, small segments of rye chromosomes were found being inserted into dif     

Genetic interactions between wheat and rye genomes in triticale

Summary Six primary triticale lines were produced from two advanced breeding lines of Triticum durum and three inbred genotypes of Secale cereale. The wheat and rye parents and the triticale derivatives were crossed in all possible combinations within each species group. Chiasma and univalent frequency of parents and hybrids were determined. The primary triticale lines had more univalents and less chiasmata per pollen mother cell than the corresponding wheat and rye parents together. The parental wheat F₁ exhibited negative heterosis for chiasma frequency whereas all rye hybrids had much higher chiasma frequencies than their inbred parents. Triticale F₁s generally showed lower chiasma frequencies and more univalents than their parents, but the degree of pairing failure was dependent upon which of the parental species within the triticale, wheat or rye, was in the heterozygous state. F₁s with heterozygous wheat genome only showed the least reduction in chiasma number (presumably caused by gene actions within the wheat genome), while F₁s with heterozygous rye genome showed high reduction in chiasma frequency and an increase in pairing failure (induced by negative interactions between the heterozygous rye and the wheat genome in triticale). A high correlation was found between the frequency of undisturbed pollen mother cells and the frequency of aneuploids in the subsequent generation. A higher number of aneuploids occurred in those populations which were heterozygous for the rye genome.     

Comparison of gas chromatography-mass spectrometry and high-performance liquid chromatography with coulometric electrode array detection for determination of alkylresorcinol metabolites in human urine

Alkylresorcinols (AR) are amphiphilic compounds present at high concentrations in the outer parts of wheat and rye kernels. Due to their specificity to whole grain and bran products of these cereals, AR and their metabolites have been proposed as biomarkers for intake of such foods. Two alkylresorcinol metabolites, 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA), have previously been quantified in human urine using two different methodologies: high-performance liquid chromatography coupled to a coulometric electrode array detector (HPLC-CEAD) and gas chromatography in combination with mass spectrometry (GC-MS). In this study, these two methodologies were compared by analysing 114 urine samples from free-living Swedish subjects consuming their habitual diet. Data were evaluated by graphical investigation of difference-plots and statistical inference of agreement was assessed by weighted Deming regression analysis. The median DHBA concentrations were 11 μM (GC-MS) and 13 μM (HPLC-CEAD), respectively. Both difference-plot and regression analysis showed a small but statistically significant additive bias, with HPLC-CEAD resulting in a slightly higher DHBA concentration than GC-MS. The median concentration of DHPPA was 18 μM for both methods. Examination of the difference-plot of DHPPA did not indicate any systematic difference between the methods, but regression analysis showed small but statistically significant constant and proportional biases. The conclusion was that the two methodologies are equally suitable for analysis of alkylresorcinol metabolites in human urine and that any small systematic differences observed are most likely of limited practical importance.