Identification of novel haze-active beer proteins by proteome analysis

Colloidal haze reduces beer quality considerably. Four haze samples were analyzed by two-dimensional gel electrophoresis (2DE) in order to identify haze-active proteins. Several protein spots were observed in all of the four haze samples. Using mass spectrometry analysis followed by a database search identified these spots as barley dimeric alpha-amylase inhibitor (BDAI-1), CMb component of tetrameric alpha-amylase inhibitor (CMb) and trypsin inhibitor CMe precursor (CMe). These proteins were considered to be haze-active. Since haze-active proteins are adsorbed by silica gel in the beer filtration process, we eluted proteins adsorbed onto silica gel (PAS) and identified their species. These major PAS were identified as protein Z4, protein Z7 and trypsin/amylase inhibitor pUP13 (TAI), rather than BDAI-1, CMb and CMe. Furthermore, we analyzed proline compositions in the beer proteins, PAS and the haze proteins. Consequently, we found that the proline compositions of PAS were higher (ca. 20 mol%) than those in the beer proteins (ca. 10 mol%), although those of the haze-active proteins such as BDAI-1, CMb and CMe were 6.6–8.7 mol%. Our results suggest that BDAI-1, CMb and CMe are not predominant haze-active proteins, but growth factors of beer colloidal haze.     

Effect of lodging on the level of mycotoxins in wheat,barley, and rice infected with the <Emphasis Type="Italic">Fusarium graminearum</Emphasis> species complex

Lodging is one possible risk factor that leads to increased cereal mycotoxin contamination, but few reports have been published on the subject. We examined the effects of lodging on the level of deoxynivalenol (DON) and nivalenol (NIV) contamination in wheat, barley, and rice infected with the Fusarium graminearum species complex. Case-control and intervention studies were applied to test the hypothesis that lodging increases the level of mycotoxin contamination. A total of 66 grain samples were collected from each field in 12 Japanese prefectures from 2002 to 2006. Each sample set consisted of grains from lodged and nonlodged plants. The concentration of DON + NIV in lodged plants was significantly higher than in nonlodged plants. All samples of wheat and barley were contaminated with DON and NIV; however, most of the lodged rice samples were contaminated only with NIV. In intervention trials to investigate the effects of lodging duration, a small area of wheat inoculated with the pathogen was completely lodged by trampling. Even with 5 days of lodging, the levels of DON + NIV in wheat grain at harvest increased by 27–51% compared to nonlodged control plots. For rice, half of each plot area was completely lodged by trampling 20 days before harvest. The level of NIV in lodged rice grain was significantly higher than that in nonlodged rice at optimum and delayed harvests, because lodging significantly increased the level of Fusarium mycotoxins in the three crops. Thus, practices (e.g., rational use of fertilizers) to avoid lodging should reduce the risk of mycotoxin contamination. This is the first epidemiological study on the effect of lodging on mycotoxin production by the F. graminearum species complex in wheat, barley, and rice.     

Evidence for bovine immunodeficiency virus infection in cattle in Zambia

We report herein on the first evidence for the presence of bovine immunodeficiency virus (BIV) in Zambia. Serological surveillance of BIV and bovine leukemia virus (BLV) was conducted in traditional cattle herds in Zambia. Out of a total of 262 sera analyzed, 11.4% were found positive for anti-BIV p26 antibodies as determined by Western blot analysis, while 5.0% were positive for anti-BLV gp51 antibodies as detected by immunodiffusion test. Peripheral blood mononuclear cells from BIV seropositive cattle were found to have BIV-provirus DNA, as detected by nested polymerase chain reaction. A nucleotide sequence corresponding to a 298 bp fragment of the BIV pol gene was also analyzed. Amino acid sequences of these Zambian pol gene products showed 98.0 to 100% homology to the American strain BIV R29, 97.0 to 99.0% to Japanese BIV isolates, and divergence ranged from 0.0 to 2.0% among Zambian BIV isolates.     

Inhibitory function of whey acidic protein in the cell-cycle progression of mouse mammary epithelial cells (EpH4/K6 cells)

Although the biological role for whey acidic protein (WAP) in milk has been suggested, its true function is not known. This paper describes evidence for WAP function in the cell-cycle progression of EpH4/K6 (EpH4), mammary epithelial cells in vitro. The forced expression of exogenous WAP significantly impaired the proliferation of EpH4 cells, whereas it did not affect that of NIH3T3 cells. Apoptosis was not enhanced in the EpH4 cells with stable expression of WAP (WAP-clonal EpH4 cells). The analyses of BrdU incorporation revealed that forced WAP expression significantly reduced incorporation of BrdU in WAP-clonal EpH4 cells compared with control cells transfected with empty plasmid. Among G1 cyclins, the level expression of cyclins D1 was significantly lower in the WAP-clonal EpH4 cells than in control cells. The inhibitory action of WAP on the proliferation of EpH4 cells was enhanced by the presence of extracellular matrix (ECM), but not by the presence of a single component comprising ECM. The cultured medium of WAP-clonal EpH4 cells inhibited the proliferation of control cells without WAP expression. The present results indicate that WAP plays a negative regulatory role in the cell-cycle progression of mammary epithelial cells through an autocrine/paracrine mechanism.     

Donor and recipient rat strains affect full-term development of one-cell zygotes cultured to morulae/blastocysts

The present study was conducted to examine the developmental potential to offspring of rat embryos cultured from 1-cell to morula/blastocyst stage. Pronuclear zygotes from Wistar x Wistar or (SD x DA) x Wistar strains were cultured in modified rat 1-cell embryo culture medium (mR1ECM) for 96 h in 5% CO(2) in air at 37 C. The proportion of the 3-way cross hybrid zygotes developing into morula/blastocyst stage (74%) was higher than that of the Wistar zygotes (66%). Day-5 morulae/blastocysts developed in vitro were transferred into Day-3 or -4 pseudopregnant recipients of Wistar or SD x DA strain. The transfer of cultured embryos resulted in the birth of offspring at 13-59%, while that of non-cultured control blastocysts showed birth rates of 35-65%. The best offspring rate of cultured embryos (59%) was obtained when the hybrid 1-cell zygotes were cultured in mR1ECM medium and transferred into the 2-days earlier uteri of SD x DA recipients. These results suggest that genetic background of recipients as well as donors is a possible factor affecting full-term development of rat morulae/blastocysts derived from 1-cell stage zygotes cultured in vitro.     

Factors affecting premature chromosome condensation of cumulus cell nuclei injected into rat oocytes

To date, production of cloned rats by somatic cell nuclear transfer (NT) has not yet been successful. Inducing premature chromosome condensation (PCC) of injected cell nuclei in recipient cytoplasm is considered essential for successful mouse cloning by the Honolulu method. In the present study, some factors affecting PCC of rat cumulus cell nuclei injected into rat oocytes were examined. Wistar female rats (young: 4 to 5-week-old, mature: > or =10-week-old) were superovulated by injections of eCG and hCG, and oocytes recovered 14 or 17 h after hCG injection were received with cumulus cell nuclei using piezo-driven micromanipulator. When the oocytes were recovered 14 h post-hCG injection from young rats and the nuclear injection into oocytes was completed within 45 min, PCC was observed in 44-49% of NT oocytes. In the case of oocytes from mature rats, PCC occurred in 11-19% of the NT oocytes. Oocytes recovered 17 h post-hCG injection did not support PCC of the injected nuclei (0-7%) regardless of the donor age. Treatment of oocytes with a neutral cysteine protease inhibitor, N-acetylleucylleucylnorleucinal, slightly increased the incidence of PCC (48 vs 37%). Comparison of rat strains for oocyte donors indicated that proportions of NT oocytes undergoing PCC in Wistar and LEW oocytes (41-46%) were higher than those in Donryu and F344 oocytes (17-25%). Thus, ability of rat oocytes to promote PCC of the injected nuclei is dependent on the characteristics of oocytes, such as age or strain of donor rats, and timing of oocyte recovery.     

Formation of a one-dimensional array of oxygen in a microporous metal-organic solid

We report the direct observation of dioxygen molecules physisorbed in the nanochannels of a microporous copper coordination polymer by the MEM (maximum entropy method)/Rietveld method, using in situ high-resolution synchrotron x-ray powder diffraction measurements. The obtained MEM electron density revealed that van der Waals dimers of physisorbed O2 locate in the middle of nanochannels and form a one-dimensional ladder structure aligned to the host channel structure. The observed O-O stretching Raman band and magnetic susceptibilities are characteristic of the confined O2 molecules in one-dimensional nanochannels of CPL-1 (coordination polymer 1 with pillared layer structure).     

A membrane-anchored protein kinase involved in Brassica self-incompatibility signaling

Self-incompatibility (SI) response in Brassica is initiated by haplotype-specific interactions between the pollen-borne ligand S locus protein 11/SCR and its stigmatic S receptor kinase, SRK. This binding induces autophosphorylation of SRK, which is then thought to trigger a signaling cascade that leads to self-pollen rejection. A recessive mutation of the modifier (m) gene eliminates the SI response in stigma. Positional cloning of M has revealed that it encodes a membrane-anchored cytoplasmic serine/threonine protein kinase, designated M locus protein kinase (MLPK). Transient expression of MLPK restores the ability of mm papilla cells to reject self-pollen, suggesting that MLPK is a positive mediator of Brassica SI signaling.