Isolation of Ciliatine (2-Aminoethylphosphonic Acid) from the Bile of the Bovine

To elucidate the formation mechanism of N,N′-dialkylpyrazine cation radical during browning reaction of sugars with amino compounds, main products in an early stage of the reaction were determined quantitatively by TLC and GLC. It was shown that the Schiff base, two-carbon fragmental product of sugar, the free radical and deoxyosone were successively produced prior to the browning. Polarographical measurements indicated that the radical formation was induced by the production of some reducing substances in the reaction mixture. These results suggest that the free radical was formed by the reduction of N,N′-dialkylpyrazinium; a compound, which demonstrated to have a strong activity to browning, might be formed by condensation of two-carbon enaminol followed by oxidation.     

DNA condensation monitoring after interaction with hoechst 33258 by atomic force microscopy and fluorescence spectroscopy

DNA condensation was only observed after the addition of Hoechst 33258 (H33258) among various types of DNA binding molecules. The morphological structural change of DNA was found to depend on the H33258 concentration. On comparison of fluorescence spectrum measurements with AFM observation, it was found that fluorescence quenching of DNA-H33258 complexes occurred after DNA condensation. Additionally, we showed that DNA condensation by H33258 was independent of sequence selectivity or binding style using two types of polynucleotides, i.e. poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Moreover, it was concluded that the condensation was caused by a strong hydrophobic interaction, because the dissolution of condensed DNA into its native form on dimethyl sulfoxide (DMSO) treatment was observed. This study is the first report, which defines the DNA condensation mechanism of H33258, showing the correlation between the single molecule scale morphology seen on AFM observation and the bulky scale morphology observed on fluorescence spectroscopy.     

Viral replication rate regulates clinical outcome and CD8 T cell responses during highly pathogenic H5N1 influenza virus infection in mice

Since the first recorded infection of humans with H5N1 viruses of avian origin in 1997, sporadic human infections continue to occur with a staggering mortality rate of >60%. Although sustained human-to-human transmission has not occurred yet, there is a growing concern that these H5N1 viruses might acquire this trait and raise the specter of a pandemic. Despite progress in deciphering viral determinants of pathogenicity, we still lack crucial information on virus/immune system interactions pertaining to severe disease and high mortality associated with human H5N1 influenza virus infections. Using two human isolates of H5N1 viruses that differ in their pathogenicity in mice, we have defined mechanistic links among the rate of viral replication, mortality, CD8 T cell responses, and immunopathology. The extreme pathogenicity of H5N1 viruses was directly linked to the ability of the virus to replicate rapidly, and swiftly attain high steady-state titers in the lungs within 48 hours after infection. The remarkably high replication rate of the highly pathogenic H5N1 virus did not prevent the induction of IFN-β or activation of CD8 T cells, but the CD8 T cell response was ineffective in controlling viral replication in the lungs and CD8 T cell deficiency did not affect viral titers or mortality. Additionally, BIM deficiency ameliorated lung pathology and inhibited T cell apoptosis without affecting survival of mice. Therefore, rapidly replicating, highly lethal H5N1 viruses could simply outpace and overwhelm the adaptive immune responses, and kill the host by direct cytopathic effects. However, therapeutic suppression of early viral replication and the associated enhancement of CD8 T cell responses improved the survival of mice following a lethal H5N1 infection. These findings suggest that suppression of early H5N1 virus replication is key to the programming of an effective host response, which has implications in treatment of this infection in humans.     

Transgenic medaka enables easy oocytes detection in live fish

Easy oocyte detection in living specimens benefits various developmental biology and environmental toxicology studies. One of the earliest markers of sex differentiation in medaka (Oryzias latipes) is oocyte development. Within the field of toxicology, a simple detection method for induced oocyte in the testis (testis-ova) as a result of endocrine disruption is necessary. In this study we produced transgenic medaka whose oocytes were labeled with fluorescent proteins using the regulatory region of the 42Sp50 gene, an isoform of polypeptide elongation 1-alpha. Short (201 nt) 5'- and 3'-flanking regions were sufficient for reporter gene expression. GFP expression was first observed in female germ cells approximately 5 days post-hatching. In the mature ovaries, germ cells showed such intense fluorescence that the fluorescence was observed from outside the body wall. In contrast, very faint fluorescence was observed in the mature testes. Testis-ova, oocytes artificially induced in the testes, were also labeled with GFP. These findings indicate through the use of transgenic medaka, that detection of female germ cells was straightforward and this transgenic medaka model proves useful for tracking female germ cells in developmental and toxicological studies.     

Pulsed-Field Gel Electrophoresis Profile Changes Resulting from Spontaneous Chromosomal Deletions in Enterohemorrhagic Escherichia coli O157:H7 during Passage in Cattle

A total of 905 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were recovered from experimentally infected cattle, in addition to the inoculated strain, were analyzed by pulsed-field gel electrophoresis (PFGE). Twelve PFGE profiles other than that of the inoculated strain were observed. We successfully identified five distinct chromosomal deletions that affected the PFGE profiles using whole-genome PCR scanning and DNA sequencing analysis. The changes in PFGE profiles of EHEC O157:H7 isolates after passage through the intestinal tract of cattle were partially generated by deletion of chromosomal regions.Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans worldwide (18). Cattle are considered the primary reservoir for this pathogen and play a central role in transmission to humans (6). Healthy cattle transiently carry EHEC O157:H7 and shed the bacteria in their feces (5, 7). Human infections have been associated with the consumption of contaminated meat and milk, direct contact with cattle, and the consumption of vegetables, fruits, and water contaminated with cattle manure (6).Because of its high discriminatory power, pulsed-field gel electrophoresis (PFGE) has been widely employed as a molecular typing method in many epidemiological investigations to identify various outbreaks and routes of transmission of EHEC O157:H7 (1, 12, 15, 17). Simpson''s index of diversity (9) was reported to be >0.985 in previous studies (1, 15), supporting the identification of richness (the number of types among isolates) and evenness (the relative distribution of individual strains among the different types) of molecular typing using PFGE.Instability of the PFGE patterns of EHEC O157:H7 isolates has been reported. Changes in PFGE patterns were observed among strains after repeated subculturing and prolonged storage at room temperature (11). Loss of Shiga toxin genes and a large-scale inversion within the genome were identified as genetic events generating changes in PFGE patterns in vitro (10, 13). Shifts in the genotypes of EHEC O157:H7 clinical isolates from patients and cattle have been reported (3, 14). This phenomenon was also observed in EHEC O157:H7 experimental infections of cattle. Spontaneous curing of a 90-kb plasmid resulted in the loss of two restricted fragments from the PFGE profiles of EHEC O157:H7 isolates obtained from experimentally infected cattle (2). The purpose of the present study was to identify the genetic events affecting the PFGE patterns of EHEC O157:H7 after passage through the intestinal tract of cattle, especially for restriction fragments that are >90 kb long.Four 5-month-old Holstein steers were housed individually in climate-controlled biosafety level 2 containment barns in accordance with the guidelines for animal experimentation defined by the National Institute of Animal Health of Japan. The pens had individual floor drains and were cleaned twice daily with water and disinfectant. All animals were healthy and culture negative for EHEC O157:H7 strains, as determined by a previously described technique (2), prior to inoculation.EHEC O157:H7 strain Sakai-215 (12, 23), which was isolated from an outbreak in Sakai, Osaka Prefecture, in 1996 was used for inoculation. This strain harbors the genes encoding Stx1 and Stx2. A spontaneous resistant strain was selected with nalidixic acid in order to facilitate the recovery of this strain from fecal samples. All calves were inoculated using a stomach tube with an exponential-phase culture (10⁹ CFU) of the nalidixic acid-resistant Sakai-215 strain. Fecal samples were collected from the four calves daily for 45 days. Fecal culturing was performed as described previously (2). Eight non-sorbitol-fermenting colonies were selected daily from each animal and identified as EHEC O157:H7 colonies by routine diagnostic methods (25).All animals were clinically normal throughout the experimental period. The EHEC O157:H7-inoculated calves (calves 1 to 4) were culture positive for the organism 24 h after inoculation. Intermittent fecal shedding by the calves was observed until 27, 32, 26, and 39 days postinoculation for calves 1, 2, 3, and 4, respectively (Fig. ​(Fig.1).1). The numbers of EHEC O157:H7 isolates recovered from calves 1, 2, 3, and 4 were 200, 224, 200, and 281, respectively.Open in a separate windowFIG. 1.Changes in PFGE profiles of EHEC O157:H7 isolates recovered from calves 1 (A), 2 (B), 3 (C), and 4 (D). The absence of a bar indicates that no EHEC O157:H7 was detected. The open horizontal bars under the vertical bars indicate that the eight isolates obtained on a day were obtained from the enrichment culture.A total of 905 recovered isolates in addition to the inoculated strain were used for PFGE analysis. Genomic DNA from each EHEC O157:H7 isolate was prepared using the method of Persing (Mayo Clinic, Rochester, MN) described by Rice et al. (20). Agarose-embedded chromosomal DNA was cleaved with XbaI by following the manufacturer''s instructions. PFGE was performed in a 0.85% megabase agarose gel, using a CHEF DR III apparatus (Bio-Rad Laboratories). The pulse time was increased from 12 to 35 s for 18 h. The PFGE profiles of all of the EHEC O157:H7 isolates recovered from the four calves were compared with that of the inoculated strain. The number of band differences was determined by enumerating the loss and addition of fragments (22).Two hundred eighty-nine isolates had PFGE profiles different from that of the inoculated strain, and 12 distinct PFGE profiles were identified for these isolates (Table ​(Table1).1). The fact that only one to three band differences were observed for the 12 profiles suggested that these isolates were closely related (22) and were variants of the inoculated strain. In addition, the pens had individual floor drains and were cleaned twice daily with water and disinfectant, which reduced the likelihood of introduction of novel EHEC O157:H7 strains. We designated the PFGE profiles A to L. PFGE profiles A, C, and H were obtained for all four calves and accounted for 30.4% of the 905 isolates recovered. Different PFGE profiles were obtained for all animals at least 2 days postinoculation (Fig. ​(Fig.1).1). All eight isolates from calf 2 collected on day 15 postinoculation and from calf 3 collected on days 22 and 23 postinoculation had PFGE profiles different from that of the original isolate (Fig. ​(Fig.1).1). The isolates that had the same PFGE profile as the inoculated strain were detected again later.

TABLE 1.

Temporal distribution of PFGE profiles of EHEC O157:H7 isolates recovered from experimentally infected cattle