Identification of a cluster of oligonucleotide repeat sequences and its practical implication in melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.) breeding

A cluster of oligonucleotide repeat sequences was identified that is linked to the melon (Cucumis melo L.) andromonoecious (a) locus, which controls andromonoecy. Six different repeat units, ranging from 11 to 49 base pairs, were clustered within a 0.6-kb intergenic region. The number of repeat units varied from two to six. After sequence analysis of diverse melon germplasms, four haplotypes of this cluster were identified. Length variations among the four haplotypes resulted from insertion or deletion of repeat sequences. Particularly, a tandem array of six repeats of 21 nucleotides was a hotspot for insertion/deletion mutations. A simple PCR-based marker was developed to identify haplotypes of this cluster based on the length polymorphism. In practice, this marker was successfully used in genetic purity tests of melon F₁ hybrid cultivars. Four self-pollinated contaminants, which were confirmed by phenotypic examination in grow-out tests, were easily discriminated from 99 F₁ hybrid individuals. In addition, the genetic distance between this marker and the andromonoecious (a) locus was calculated as 7.9 cM, after analyzing melon F₂ populations originating from a cross between monoecious and andromonoecious parental lines. Therefore, this marker will be useful as a recombinant selection marker in marker-assisted backcrossing of monoecy in melon breeding programs.     

Human metabolism of atrazine

Atrazine (ATZ) metabolism by human liver microsomes (HLM), cytochrome P450 (CYP) isoforms, and human liver (HL) S9 fractions, was investigated using HPLC/PDA and LC/MS/MS. CYP-dependent metabolites from pooled HLM are desethylatrazine (DEA), desisopropylatrazine (DIA), 1-hydroxyisopropylatrazine (HIATZ), and 2-hydroxyethyl atrazine (HEATZ). DEA and DIA were major metabolites in pooled HLM. CYP1A2 and 2C19, respectively, were major isoforms for DEA and DIA production. CYP3A4, while less active, is generally at high concentrations, produces both DEA and DIA and is significant. The percent total normalized rates (%TNR) for CYP1A2 and 3A4 in pooled HLM were 63% and 24% for DEA, and 35% and 56% for DIA production. Single donor HLM samples, showed correlations for CYP1A2 (r = 0.92) and 3A4 (r = 0.81) for DEA and DIA production, while variations in production of DEA and DIA were 8.5- and 6.0-fold, respectively. Pooled S9 fractions also mediate glutathione conjugation of atrazine, DEA and DIA.     

Flowering and changes in respiration in Asiatic hybrid lilies as influenced by bulb vernalization

Asiatic hybrid lilies, Lilium × elegans Thunb., ‘Red Carpet’ and ‘Sunray’ were used to investigate the effect of bulb vernalization at 2.5 °C on plant growth, flowering, and CO₂ production (respiration), and to use the CO₂ production pattern to monitor the time of flower bud initiation and development. Lily shoot emergence and flowering were accelerated when bulbs received 2.5 °C bulb vernalization; however, flowering was delayed when bulbs were stored at 20 °C before treatment at 2.5 °C; this indicated that bulbs were de-vernalized. The maximum CO₂ level, and the minimum level, reached in 78 h in non-vernalized bulbs and in 110 h in 6 weeks of 2.5 °C (6 weeks/2.5 °C) treated bulbs, was increased as the 2.5 °C duration was increased; this indicated that CO₂ level can be an useful parameter to measure the cold stimulus (i) accumulated in bulbs following bulb vernalization. The respiration rate higher than the predicted values of the best-fit curves derived from the quadratic equations was designated as Blip A and this was correlated to the time of flower bud initiation and development. Shoot elongation may follow the rise in carbon dioxide levels after reaching the minimum level. It is proposed that increased carbon dioxide levels higher than the predicted levels (Blip A), was correlated to the time of flower bud initiation and development. Measurement of carbon dioxide production upon receipt of bulbs may be a useful technique to provide important information for optimum vernalization treatments for bulbs that have accumulated different levels of low temperature stimulus after bulb vernalization.     

Promising antidiabetic potential of fucoxanthin isolated from the edible brown algae Eisenia bicyclis and Undaria pinnatifida

In the present study, we investigated the antidiabetic potential of fucoxanthin via the inhibition of rat lens aldose reductase (RLAR), human recombinant aldose reductase (HRAR), advanced glycation end-product (AGE) formation, protein tyrosine phosphatase 1B (PTP1B), and α-glucosidase. Fucoxanthin displayed potent inhibitory activity against AGE formation and HRAR and RLAR activity. In addition, fucoxanthin showed potent inhibitory activity against PTP1B. However, it did not show α-glucosidase inhibitory activity below 200 μM. In addition, our kinetic study revealed that fucoxanthin competitively inhibited RLAR, while it showed mixed-type inhibition against PTP1B. In order to confirm enzyme inhibition, we predicted the 3D structure of PTP1B using Autodock 4.0 to simulate the binding of fucoxanthin. Docking simulation results demonstrated that three residues of PTP1B (Phe30, Phe52, and Gly183) interact with the two hydroxyl groups of fucoxanthin. In addition, the binding energy was negative (?7.66 kcal/mol), indicating that the three hydrogen bonds may stabilize the open form of the enzyme and potentiate tighter binding to the active site of PTP1B, resulting in more effective PTP1B inhibition. The results of the present study therefore clearly demonstrate the promising potential of fucoxanthin as a therapeutic intervention for the management of diabetes as well as diabetes-associated complications, which could be explored further.     

Quercetin-3-O-β-d-glucuronopyranoside (QGC)-induced HO-1 expression through ERK and PI3K activation in cultured feline esophageal epithelial cells

Heme oxygenase-1 (HO-1) is one of the antioxidant enzymes which help protect against cellular damage. The present study examined the ability of Quercetin-3-O-β-d-glucuronopyranoside (QGC), flavonoid glucoside extracted from Rumex Aquaticus Herba, to induce expression of HO-1 and analyzed its signaling mechanism in cultured feline esophageal epithelial cells (EEC). Culture of the esophageal epithelial cells from cat was prepared. The data suggested that QGC could result in enhanced antioxidant enzyme defense system via HO-1 expression and Nrf2 translocation involving both the ERK and PI3K-Akt pathways as well as partly PKC pathways in EEC.     

Prevalence of genes for enterotoxins,toxic shock syndrome toxin 1 and exfoliative toxin among clinical isolates of Staphylococcus pseudintermedius from canine origin

A total of 74 Staphylococcus pseudintermedius strains were isolated from the 99 clinical cases of canine pyoderma or chronic otitis in our veterinary teaching hospital during May 2006–February 2008. In this study, we examined the genetic distribution of staphylococcal pyogenic toxins such as staphylococcal enterotoxins A (sea), B (seb), C (sec), D (sed), E (see), and toxic shock syndrome toxin 1 (tst) as well as the previously characterized S. intermedius exfoliative toxin (siet) among those isolates. The polymerase chain reaction analyses with the toxin gene‐specific primers revealed that 18 (24.3%) of 74 S. pseudintermedius isolates carried the sec genes, but none of the sea, seb, sed, see and tst genes. Further DNA sequencing analysis of the amplified sec genes revealed that they all belonged to the canine type C staphylococcal enterotoxin (SEC_canine) whose superantigenic activity has been demonstrated. In addition to the sec_canine genes, our polymerase chain reaction results showed that all the 74 isolates carried the siet gene. Since both SEC_canine and SIET toxins are known to be biologically active, it would be interesting to investigate how those toxins are involved in the pathogenesis of the canine diseases by S. pseudintermedius such as pyoderma or chronic otitis.     

Evaluation of haplotypes associated with copper toxicosis in Bedlington Terriers in Australia

OBJECTIVE: To evaluate the haplotype distribution associated with the copper toxicosis gene and the segregation of the mutated allele in a Bedlington Terrier population in Australia. ANIMALS: 131 Bedlington Terriers. PROCEDURE: Samples of DNA and RNA were obtained from each dog. Genetic status of each dog was evaluated by use of the DNA markers C04107; single nucleotide polymorphism (SNP), which was adjacent to exon 2 of Murr1; and a deletion marker for exon 2. A subgroup of the study population was evaluated by use of biochemical and histologic techniques to elucidate the correlation between genotype and phenotype. RESULTS: We identified a recombination between the C04107 marker and Murr1 and a variation in a nucleotide in the splice site of exon 2 in our Bedlington Terrier cohort. Furthermore, we identified a novel haplotype associated with copper toxicosis in this cohort. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings indicate that the deletion of exon 2 was not the sole cause of copper toxicosis, although only exon 2 deletion of Murr1 has been responsible for copper toxicosis in Bedlington Terriers. Although we failed to find a novel mutation in our cohort, we identified an affected dog family with an intact exon 2. Furthermore, we found that an SNP in the 5' splicing site of exon 2 may or may not be associated with a novel mutation of the Murr1 gene or other genes. Loss of linkage between the C04107 marker and the Murr1 gene was also identified in a certain family of dogs.     

Recruit of porcine oocytes excluded from nuclear transfer program for the production of embryos following parthenogenetic activation

To evaluate whether oocytes excluded from somatic cell nuclear transfer (SCNT) could be utilized for embryo production by parthenogenetic activation (PA), porcine oocytes with poor morphology after maturation culture were excluded from SCNT and subsequently used for PA with different stimuli. In the first set of experiment, either electric pulse of different strengths (1.75, 2.0 or 2.25 kV/cm for 30 microsec each) or chemicals with different treatment durations [7% ethanol for 5 min followed by exposure to 6-dimethylaminopurine (6-DMAP) for 0, 2, 3 or 4 hr] was employed. Development to the 8-cell and morula stages was significantly (P<0.05) improved by electric stimulation of 2.0 kV/cm, while blastocyst formation was enhanced by chemical treatment of ethanol and 6-DMAP for 4 hr. Subsequently, oocytes were parthenogenetically activated by one of four stimuli; 1) optimal electric (2.0 kV/cm for 30 microsec), 2) optimal chemical (ethanol followed by 6-DMAP for 4 hr), 3) electric then chemical and 4) vice versa. On the other hand, oocytes with normal morphology were subjected to the same experimental treatments for the control. Regardless of oocyte type, a combination of electric and chemical stimulations did not further stimulate preimplantation development, compared with electric activation only. However, combinational treatment greatly increased the cell number of blastocysts in SCNT-excluded oocytes (21.9 to 22.9 vs. 16.9 cells/blastocyst), while such effect was not found in normal oocytes (22.2 to 23.3 cells/blastocyst). In conclusion, porcine oocytes excluded from SCNT still have a potential to develop blastocysts after PA and this might contribute to increasing the efficiency of SCNT for various purposes. A combined activation by electricity and chemical yielded the best rate of preimplantation development with increasing the quality of blastocyst.     

A blocking ELISA with improved sensitivity for the detection of passively acquired maternal antibodies to BHV-1

Four serological tests were evaluated for their ability to detect passively acquired maternal antibodies to Bovine herpesvirus 1. A blocking enzyme-linked immunosorbent assay demonstrated superior sensitivity in the detection of such antibodies in calves up to 9-11 months old, versus calves up to 7 months old for other tests.     

Relationship between DNA ploidy and proliferative cell nuclear antigen index in canine hemangiopericytoma.

The mitotic index is reported to be correlated with recurrence, mean patient survival, and metastasis of canine hemangiopericytoma (CHP). However, to the authors' knowledge, studies investigating the parameters that can predict recurrence or metastasis of CHP with low mitotic index have not been done. To evaluate growth kinetics of CHP with low mitotic index, a retrospective analysis of the proliferative activity by antiproliferative cell nuclear antigen monoclonal antibody and DNA contents by flow cytometry (FCM) was performed with 21 formalin-fixed and paraffin-embedded CHP samples. Of the 21 tumors evaluated by FCM, 6 (26.6%) were aneuploid tumors, and 15 (71.4%) were diploid tumors. There was significant correlation between the PCNA index and ploidy pattern. The diploid group had 39.1 +/- 9.2 PCNA index, whereas the aneuploid group's proliferative cell nuclear antigen (PCNA) index was 63.1 +/- 8.2. The diploid group had mean mitotic index value of 1.140 +/- 0.855, and the aneuploid group had a mean value of 1.067 +/- 0.767. From these results, the CHP samples with low mitotic index were classified into either the aneuploid group with higher PCNA index or the diploid group with lower PCNA index, suggesting that DNA ploidy and proliferative activity may give an indication about malignancy of CHPs with a low mitotic index.