Engineering of a broad specificity antibody for simultaneous detection of 13 sulfonamides at the maximum residue level

Sulfa antibiotics (sulfonamides) are a group of molecules sharing the p-aminobenzenesulfonamide moiety. Sulfonamides are used in veterinary and human medicine. Sometimes, the meat or milk of medicated animals is contaminated with residual sulfonamides. Current analytical methods for sulfonamides are unfit for screening of food, because they are either too laborious, insensitive, or specific for a few sulfa compounds only. A rapid immunoassay for detection of all sulfas in a single reaction would thus be useful. Previously, we used protein engineering to improve the broad specificity of sulfa antibody 27G3. In this study, we improved the best mutant of the previous studies with site-directed mutagenesis. The new mutants recognized different sulfonamides with affinities sufficient for detection of all 13 tested sulfonamides below the MRL level. We furthermore demonstrated the functionality of one mutant in some real sample matrices.     

Areal activities and stratification of hydrolytic enzymes involved in the biochemical cycles of carbon, nitrogen, sulphur and phosphorus in podsolized boreal forest soils

A novel approach allowing on-site high throughput enzyme activity measurements by fluorogenic model substrates was applied to study the functioning of enzymes involved in biochemical cycling of nutrients in boreal forest soil ecosystems. The examined enzymes comprised α-glucosidase, β-glucosidase, β-xylosidase, β-cellobiosidase, N-acetyl-glucosamidase, acetate-esterase, butyrate-esterase, phosphomonoesterase, sulphatase and aminopeptidase, whereby spatial and seasonal variation of their activity was investigated over nine seasons in 2 years. The studied sites of boreal podzolized soil of Pinus sylvestris and Picea abies forest were located in central Finland. Activity of all enzymes except sulphatase was highest in the humus layer in all seasons. Maximum sulphatase activity was located below the humus layer in the soil column. Annual activities of acetate-esterase, butyrate-esterase, β-glucosidase and phosphomonosterase calculated to in situ temperature during the year were 480-700, 690-950, 110-190 and 110-200 mol m⁻² year⁻¹, respectively. They were up to 100 fold higher than the other six measured activities. The overall turnover capacity of the enzymes was >1000 mol of ester linked carbon, >700 mols carbon from different carbohydrates, 100-200 mol of ester linked phosphate, 10-40 mol of ester linked sulphate m⁻² year⁻¹. Winter time (November-April) contributed from 7 to 32% to the annual turnover capacity indicating important enzyme activities also during a cold period of the year. Clear-cutting of the tree stand did not adversely affect enzyme activities related to the cycling of carbon, nitrogen, sulphur and phosphorus during the year. The pH optimum for hemicellulose and cellulose hydrolysing enzymes was pH 3-4 and the pH optimum of phosphomonoesterase, sulphatase, aminopeptidase and N-acetyl-glucosamidase was 4-5. This shows that the hydrolytic activities were adapted to the acid pH-values of the soils. The soil hydrolytic potential was many fold higher as compared to the actual amount of litter it received in the P. sylvestris and P. abies forests.     

Identification and validation of a quantitative trait locus associated with wheat yellow mosaic virus pathotype I resistance in a Japanese wheat variety

Yellow mosaic disease, caused by wheat yellow mosaic virus (WYMV), is one of the most serious diseases of winter wheat (Triticum aestivum L.) in Japan. The three pathotypes of WYMV are distributed in different geographical areas: pathotype I is found mainly in western and central Japan (Kanto), pathotype II in northern Japan (Tohoku and Hokkaido) and pathotype III on the southern island of Japan (Kyushu). A total of 246 doubled‐haploid (DH) lines, derived from a cross between ‘Yumechikara’ (resistant) and ‘Kitahonami’ (susceptible), were evaluated for 2 years for their resistance to WYMV pathotype I. A single major quantitative trait locus, Q.Ymym, mapping to chromosome 2D was associated with resistance to pathotype I in ‘Yumechikara’. This is the first time a QTL responsible for pathotype I resistance has been identified. Fine mapping of Q.Ymym indicated that it was on a tight linkage block originating from ‘Yumechikara’, and the markers associated with this block will accelerate the development of varieties resistant to WYMV pathotype I.     

Blue aleurone trait diagnoses developmental origin of three pistils in a floret in common wheat

The common ‘three‐pistil’ (TP) wheat mutation line expresses TPs in a floret normally containing TPs forming three grains set close back‐to‐back. The developmental origin of the TP trait in common wheat had been diagnosed non‐destructively using the blue aleurone trait. The aleurone colour of F₂ seeds grown in F₁ plants of cross TP/UC66049 was evaluated. Due to xenia, the hue of blue grain colour depended on dose of the Ba1 gene for blue aleurone in the triploid endosperm. The TP trait produced four types of segregation in three‐seed clusters: (i) white grain only, (ii) two white grains and one blue, (iii) one white grain and two blue, and (iv) three blue grains only. The observed frequency of blue–white seed within clusters followed the binominal distribution ₃C_r (0.75)^r·(0.25)^3–r, where r is the number of colour variants in three‐seed clusters (r = 0–3). Intrafloret segregation of seed colour and F₂ segregation derived from aleurone colour of F₃ seeds indicated an independent origin of the TP trait.     

The Dnmt3b splice variant is specifically expressed in in vitro-manipulated blastocysts and their derivative ES cells

Manipulation of preimplantation embryos in vitro, such as in vitro fertilization (IVF), in vitro culture (IVC), intracytoplasmic sperm injection (ICSI), somatic cell nuclear transfer (SCNT) and other assisted reproduction technologies (ART), has contributed to the development of infertility treatment and new animal reproduction methods. However, such embryos often exhibit abnormal DNA methylation patterns in imprinted genes and centromeric satellite repeats. These DNA methylation patterns are established and maintained by three DNA methyltransferases: Dnmt1, Dnmt3a and Dnmt3b. Dnmt3b is responsible for the creation of methylation patterns during the early stage of embryogenesis and consists of many alternative splice variants that affect methylation activity; nevertheless, the roles of these variants have not yet been identified. In this study, we found an alternatively spliced variant of Dnmt3b lacking exon 6 (Dnmt3bΔ6) that is specific to mouse IVC embryos. Dnmt3bΔ6 also showed prominent expression in embryonic stem (ES) cells derived from in vitro manipulated embryos. Interestingly, IVC blastocysts were hypomethylated in centromeric satellite repeat regions that could be susceptible to methylation by Dnmt3b. In vitro methylation activity assays showed that Dnmt3bΔ6 had lower activity than normal Dnmt3b. Our findings suggest that Dnmt3bΔ6 could induce a hypomethylation status especially in in vitro manipulated embryos.     

Proteome analysis of whole and water‐soluble proteins in masseter and semitendinosus muscles of Holstein cows

To assess both quantitative and qualitative differences between the slow‐ and fast‐type muscles, masseter (slow) and semitendinosus (fast) from four Holstein cows were analyzed by two‐dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry. The proteome analysis identified 27 spots as 20 proteins in the whole protein fraction extracted with 8 mol/L urea solution, and 16 spots were identified as 11 proteins in the water‐soluble protein fraction. Two slow‐type myofibrillar proteins (myosin light chain‐1 slow‐b and myosin light chain‐2 slow), and aconitase‐2 mitochondria were present at higher levels in the masseter muscle (P < 0.05). Four fast‐type myofibrillar proteins (myosin light chain‐1 fast, myosin light chain‐2 fast, myosin light chain‐3 fast and tropomyosin‐1), and three enzymes of glycolytic pathway (enolase‐3, aldolase‐A and triosephosphate isomerase), were present at higher levels in the semitendinosus muscle (P < 0.05). Our proteome analysis showed that the composition of sarcoplasmic proteins as well as myofibrillar proteins was clearly different between slow‐ and fast‐type muscles.     

Characterization of local rhizobia in Thailand and distribution of malic enzymes

TWenty-six isolates were obtained from nodules of various legume plants (Glycine max, Vigna sinensis, Arachis hypogaea, Desmanthus virgatus, Acacia mangium, Centrosema pascuorum, Pterocarpus indicus, Xylia xylocarpa, and Sesbania rostrata) in Thailand. After confirming their nodulation and nitrogen-fixing abilities, they were identified by 16S rRNA gene analysis as Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium leguminosarum, Rhizobium gallicum, and Rhizobium galegae. Using these local isolates, the distribution of the activities of both NAD⁺-dependent (DME: EC 1.1.1.39) and NADP⁺-dependent (TME: EC 1.1.1.40) malic enzymes was surveyed. The malic enzyme activities were present in all the isolated rhizobia and in other 17 local Bradyrhizobium strains in Thailand. In almost all the rhizobia, the DME activity predominated whereas the TME activity predominated only in the Rhizobium gallicum strains that were major symbionts of Sesbania rostrata. Southern hybridization analysis was performed to survey the distribution of the malic enzyme genes among the local rhizobia, which are similar to those of B. japonicum. DNA probes (ME1 for DME and ME2 for TME) were prepared by polymerase chain reaction (PCR) using degenerated primers from conserved regions of the protein sequences of bacterial malic enzymes. Southern blot analysis with ME1 as a probe showed a single band in about half of the isolates, especially in B. japonicum and R. leguminosarum strains, suggesting the wide distribution of such DME genes among local rhizobia. In contrast, Southern blot analysis with ME2 as a probe detected a single band only in five B. japonicum strains, suggesting that the TME genes, which are similar to those of B. japonicum, would be unique in a group of B. japonicum.     

Development of an enzyme-linked immunosorbent assay (ELISA) for residue analysis of the fungicide azoxystrobin in agricultural products

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for residue analysis of azoxystrobin in garden crops, for which the maximum residue limits (MRLs) are 0.5-50 mg/kg in Japan. For hapten synthesis, an ethyl carboxyl group was introduced to the 4-position of the 2-cyanophenoxy group in azoxystrobin, and its cyano group was changed to a methyl group. An anti-azoxystrobin monoclonal antibody was prepared from mice immunized with hapten-keyhole limpet hemocyanin conjugate. The dc-ELISA using prepared antibody showed 50-250-fold higher sensitivity compared to the MRLs. The working range of the dc-ELISA was 10-200 ng/mL. The dc-ELISA showed high specificity to azoxystrobin. When methanol extracts from nine kinds of garden crops spiked with azoxystrobin ranging near the MRLs were analyzed, the determined results by the dc-ELISA agreed well with the results of their controls. In addition, azoxystrobin spiked in garden crops homogenates was satisfactorily extracted by methanol solution and easily analyzed. The recovery rate of dc-ELISA was 96-109% and correlated well with the results obtained by HPLC analysis.     

Waxy wheat as a functional food for human consumption

Waxy wheat (Triticum aestivum L.), a type of wheat that was first developed in Japan, produces grain amylopectin. I carried out three researches. Firstly, using Japanese adults, we carried out sensory evaluation of three types of mochi (a dumpling-like cake made of steamed and pounded grains) prepared from waxy wheat, normal wheat and waxy rice, and demonstrated that waxy wheat mochi had low adhesiveness and had the greatest ease of swallowing among the samples tested. Secondly, using the members of the JSDR (the Japanese Society of Dysphagia Rehabilitation) who attended the annual meeting, we performed another texture evaluations between waxy wheat and waxy rice mochi. The respondents reported waxy wheat mochi was less sticky and thus easier to chew and swallow than waxy rice mochi.