Comparison of protein chemical and physicochemical properties of rapeseed cruciferin with those of soybean glycinin

Rapeseeds contain cruciferin (11S globulin), napin (2S albumin), and oleosin (oil body protein) as major seed proteins. The effects of oil expression and drying conditions on the extraction of these proteins from rapeseed meal were examined. The conditions strongly affected the extraction of oleosin and only weakly affected the extraction of cruciferin and napin. The protein chemical and physicochemical properties of cruciferin, the major protein present, were compared with those of glycinin (soybean 11S globulin) under various conditions. In general, cruciferin exhibited higher surface hydrophobicity, lower thermal stability, and lower and higher solubility at mu= 0.5 and mu = 0.08, respectively, than did glycinin. At the pHs (6.0, 7.6, and 9.0) and ionic strengths (mu= 0.08 and 0.5) examined, the emulsifying ability of cruciferin was worse than that of glycinin, except at mu= 0.08 and pH 7.6. The emulsifying abilities of cruciferin and glycinin did not correlate with thermal stability and surface hydrophobicity. Higher protein concentration, higher heating temperature, higher pH, and lower ionic strength were observed to produce harder gels from cruciferin. Gel hardness partly correlated with the structural stability of cruciferin.     

Simple and rapid determination of thiabendazole,imazalil, and o-phenylphenol in citrus fruit using flow-injection electrospray ionization tandem mass spectrometry

A simple and rapid analytical method for thiabendazole (TBZ), imazalil (IMA), and o-phenylphenol (OPP) in citrus fruit has been developed by using flow-injection electrospray ionization tandem mass spectrometry for the first time. The method involves the combined use of stable isotopically labeled internal standards (thiabendazole-(13)C(6), imazalil-d(5), and p-phenylphenol-d(9)) and a multiple reaction monitoring technique. The average recoveries for the fungicides at the tolerance levels (TBZ and OPP, 10 mg/kg; IMA, 5 mg/kg) ranged from 77 to 101%, with the coefficients of variation (CVs) ranging from 0.7 to 4.2% (n = 5). At half the tolerance levels (TBZ and OPP, 5 mg/kg; IMA, 2.5 mg/kg), the average recoveries ranged from 62 to 112%, with the CVs ranging from 0.7 to 8.4% (n = 5). The CVs of the average recoveries, obtained from lemon samples fortified with three fungicides at the tolerance levels, obtained on three different days over two weeks, ranged within 2%. The analysis time, including sample preparation and determination, is only 15 min.     

Distribution and Fate of Organotin Compounds in Japanese Coastal Waters

In order to elucidate the details of both the distribution and fate of organotin compounds (OTs) in the costal ecosystem, the concentrations of butyltin compounds (BTs) and phenyltin compounds (PTs) were determined in seawater, sediment and blue mussels Mytilus galloprovincialis collected in Maizuru Bay, Japan. The concentrations of tributyltin (TBT) in seawater, sediments and mussels ranged from 3.9 to 27 ng l^?1, from 1.2 to 19 ng g^?1 dry wt and from 0.77 to 11 ng g^?1 wet wt, respectively. Although the levels of TBT in seawater, sediments and mussels from Maizuru Bay were lower than those reported previously at other sites in Japan, the levels can still be toxic to susceptible organisms. Trace amounts of PTs were also found in seawater, sediment and mussel samples, indicating that there is a slight input of triphenyltin (TPT) into the seawater in the bay at present. The highest TBT concentration in seawater among all sites measured in the bay was found near a glass factory, and the lowest concentrations were observed at the center of the bay. The highest concentration of TBT in sediment was detected near a shipyard. In mussels, a high concentration of TBT was detected near a cement plant and timberyard. These results indicate that the major sources of contamination of OTs in the bay are considered to be from those facilities and ships. The proportion of TBT in seawater and mussels showed that, of total BTs, it was the predominant compound at most sites. These results suggested that there is a continuous input of TBT into the seawater and thereafter an accumulation of TBT in mussels due to their low metabolic capacity to degrade TBT. In sediment, the proportion of monobutyltin (MBT) was the highest of BTs at most sites. This could reflect a previous contamination by TBT used before the regulation of its usage in the bay.     

Screening of fungal strains responsible for strong fungistasis against Fusarium oxysporum f. sp. radicis-lycopersici in a coffee compost-amended soil

(pp. 817–824)
Disease incidence of crown and root rot of tomato, caused by Fusarium oxysporum f. sp. radicis-lycopersici J3 (FOL J3) was significantly lower in a soil (CC-soil) amended with coffee compost and chemical fertilizers (CF) than in a soil (CF-soil) amended with only CF. Germination of microconidia of various plant-pathogenic Fusarium oxysporum strains was consistently lower in the CC-soil than in the CF-soil, suggesting that the CC-soil possessed a higher degree of fungistasis. When the CC-soil was supplemented with rifampicin and kanamycin, germination of FOL J3 didn't increase, suggesting that the higher degree of fungistasis in the CC-soil may not be of bacterial origin. The substrate-induced respiration inhibition method demonstrated that the CC-soil possessed higher microbial activity and was dominated by fungi. PCR-DGGE analysis showed that the microbial community structure of the two soils was different. Fungal mycelia were isolated from the soils and the effect of the isolates on soil fungistasis was examined. Three isolates, all belonging to F. oxysporum out of 49 showed strongest suppressive effect on the germination of FOL J3 and two isolates suppressed Fusarium crown and root rot disease when they were inoculated into autoclaved CC-soil. These results might suggest that the isolates close to F. oxysporum were responsible for highly fungistatic capability in the CC-soil and were a possible source for disease suppression.     

Evaluation of the solubility and emulsifying property of soybean proglycinin and rapeseed procruciferin in relation to structure modified by protein engineering

The presence or absence of a highly negatively charged extension region in beta-conglycinin (J. Agric. Food Chem. 1999, 47, 5278) and the length of a highly negatively charged variable region IV in glycinin (J. Agric. Food Chem. 2004, 52, 8197) are important determinants of solubility and emulsifying property. To examine the effects of the variable region IV from proglycinin A1aB1b and A3B4 and of the extension region from beta-conglycinin alpha' (alpha'ext) on solubility and emulsifying properties in detail, several mutants of proglycinin, procruciferin, and beta-conglycinin were designed and prepared in Escherichia coli. Nine out of 10 mutants were expressed at high levels in E. coli and shown to be homotrimer similar to the wild types as assessed by gel filtration. The position of the introduced negatively charged region as well as the amino acid composition were demonstrated to affect solubility at mu = 0.08. All of the proglycinin, procruciferin, and beta-conglycinin mutants with the alpha'ext in the C-terminus, especially the proglycinin mutant, exhibited excellent emulsifying ability and emulsion stability. These indicate that improvement of emulsifying properties by insertion of the alpha'ext in the C-terminus may be generally applicable to seed globulins.     

Structure-physicochemical function relationships of soybean glycinin at subunit levels assessed by using mutant lines

Glycinin is a hexameric protein composed of five kinds of subunits. The subunits are classified into two groups, group I (A1aB1b, A1bB2, and A2B1a) and group II (A3B4 and A5A4B3). We purified four mutant glycinins composed of only group I subunits (group I-glycinin), only group II subunits (group II-glycinin), only A3B4 (A3B4-glycinin), and only A5A4B3 (A5A4B3-glycinin) from mutant soybean lines. The physicochemical properties of these glycinin samples were compared with those of the normal glycinin (11S) composed of five kinds of subunits. The thermal stabilities (as measured by thermal denaturation midpoint temperatures) of 11S, group I-glycinin, and group II-glycinin were similar to each other, although that of A3B4-glycinin was significantly lower than those of the others. The orders of aromatic and aliphatic surface hydrophobicities were the same: A3B4-glycinin > group II-glycinin > A5A4B3-glycinin > 11S > group I-glycinin. The solubility of 11S as a function of pH at mu = 0.5 was governed by that of group I-glycinin and followed this order at acidic pH: 11S = group I-glycinin > A3B4-glycinin > group II-glycinin = A5A4B3-glycinin. The order of emulsifying abilities was A5A4B3-glycinin > group II-glycinin > A3B4-glycinin > 11S > group I-glycinin. This order was consistent with that of the length of their hypervariable regions. Except for this relationship, there was no significant relationship among the other physicochemical properties of the mutant glycinins.     

Improvement of a rapid diagnosis kit to detect either influenza A or B virus infections

To improve the sensitivity of a kit, ESPLINE INFLUENZA A&B for rapid diagnosis of influenza by detecting influenza A or B virus specific nucleoproteins (NP), the ESPLINE INFLUENZA A&B-N was developed by using newly established monoclonal antibodies (MAbs) to the respective NP molecule. MAbs FVA2-11 and FrB1-03 recognize the epitope on the amino acid region 59-130aa of the NP molecule of influenza A virus, and that on the region 72-191aa of the NP of influenza B virus, respectively. The new kit detected influenza A and B virus antigens with a detection limit of 10(2.0)-10 (2.7) pfu/test, which is 4-1000 times higher than that of the original kit. Importantly, this kit detected each of influenza A viruses of the known hemagglutinin (HA) subtypes (H1-H15) including the H5N1 viruses recently isolated from human and avian sources in Asia. The kit also detected all of the 15 representative influenza B virus strains tested. The ESPLINE INFLUENZA A&B-N is thus a rapid and highly sensitive and specific kit for the diagnosis of either influenza A or B virus infections.     

Characterization and crystallography of recombinant 7S globulins of Adzuki bean and structure-function relationships with 7S globulins of various crops

The recombinant proteins Adzuki 7S1, Adzuki 7S2, and Adzuki 7S3 were prepared through the Escherichia coli expression systems of three kinds of adzuki bean cDNAs. The recombinant proteins exhibited intrinsic thermal stabilities, surface hydrophobicities, and solubilities, although the homology of their amino acid sequences ranged from 95-98%. To understand why these individual proteins exhibited different properties, their three-dimensional structures were elucidated. The three proteins were successfully crystallized, and the three-dimensional structures of Adzuki 7S1 and Adzuki 7S3 were determined. The properties and structures of these two proteins were comprehensively compared with those of recombinant 7S globulins (soybean beta-conglycinins beta and alpha'c and mungbean 8Salpha) reported previously. It was likely that cavity sizes, hydrogen bonds, salt bridges, hydrophobic interactions, and lengths of loops determine the thermal stabilities of 7S globulins, and results indicated that cavity sizes strongly contribute to such stability. Surface hydrophobicity was also found to be determined not only by distributions of hydrophobic residues on the molecular surface. Furthermore, solubility at neutral and weak alkaline pH values at mu = 0.08 was found to be dominantly influenced by the electrostatic surface potentials.     

Testicular compensatory hypertrophy related to hemicastration in prepubertal dogs

Several studies have reported that the plasma testosterone (T) level and semen volume are compensated after hemicastration (HEC) in adult dogs, but that the sperm count is not. Nevertheless, the effects of HEC in prepubertal dogs have not been reported. In this study, HEC was performed at 16 weeks of age in 8 male beagles, and the function of the residual testis was investigated until 48 weeks of age. The testis volume was consistently higher in the HEC group than the control (CON) group. After 40 weeks of age, compensatory hypertrophy was observed, with a mean testis volume of 125% of that in the CON group (p<0.05). Furthermore, the semen volume and plasma testosterone (T) level were similar to those in the CON group, suggesting functional compensation, but the sperm count was not compensated. These results showed that the testis volume, semen volume, and plasma T level were compensated after HEC in prepubertal dogs, but the spermatogenic function was not.