Selective growth-inhibiting effects of compounds identified in Tabebuia impetiginosa inner bark on human intestinal bacteria

The growth-inhibiting activity of anthraquinone-2-carboxylic acid and lapachol identified in the inner bark of taheebo, Tabebuia impetiginosa, toward 10 human intestinal bacteria was evaluated by using a paper disk diffusion bioassay and compared to those of seven lapachol congeners (1,4-naphthoquinone, naphthazarin, menadione, lawsone, plumbagin, juglone, and dichlone) as well as two commercially available antibiotics, chloramphenicol and tetracycline. Anthraquinone-2-carboxylic acid exhibited very strong growth inhibition of Clostridium paraputrificum at 1 microg/disk while 100 microg/disk of lapachol was needed for moderate growth inhibition of the same organism. These two isolates exhibited weak inhibition of Clostridium perfringens and Escherichia coli at 100 microg/disk while no adverse effects were observed on the growth of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Lactobacillus acidophilus, and Lactobacillus casei at 1000 microg/disk. Structure-activity relationships indicate that a methyl group in the C-2 position of 1,4-naphthoquinone derivatives might play an important role in antibacterial activity.     

Phylogeographic patterns of Armillariaostoyae in the western United States

Nuclear ribosomal DNA regions (i.e. large subunit, internal transcribed spacer, 5.8S and intergenic spacer) were sequenced using a direct‐polymerase chain reaction method from Armillaria ostoyae genets collected from the western USA. Many of the A. ostoyae genets contained heterogeneity among rDNA repeats, indicating intragenomic variation and likely intraspecific hybridization. Intragenomic variation was verified by visually editing base‐sequence offsets in regions with insertions/deletions, and using sequence‐specific internal primers to resequence heterogeneous regions. Phylogenetic analyses with Bayesian Inference methods were used to define groups within A. ostoyae. Analysis of A. ostoyae from outside the western USA indicated the presence of a Circumboreal group of A. ostoyae that also occurs in Utah; two other phylogeographic groups were associated with the Rocky Mountain and Pacific Northwest regions of the USA. Mixed sequence types, an indication of intraspecific hybrids, were common in some geographic regions. Hybridization events may have influenced species evolution, contributing to variation in pathogenicity and virulence. The occurrence of these groups and intraspecific hybrids also indicates that paleogeography and paleoclimate may have influenced the phylogeography of A. ostoyae. In addition, other Armillaria species were examined for evolutionary relationships with the groups of A. ostoyae. These findings will provide a basis for future research relating ecological function to genetic diversity within A. ostoyae.     

The effects of enrofloxacin on canine tendon cells and chondrocytes proliferation <Emphasis Type="Italic">in vitro</Emphasis>

Enrofloxacin, a fluoroquinolone antibiotic has been used widely in humans and domestic animals, including dogs, because of its broad-spectrum activity and relative safety. The side effects of fluoroquinolone, induced tendinopathy, tendonitis, spontaneous tendon rupture and cartilage damage, remain incompletely understood. In the present study, we investigated the in vitro effects of enrofloxacin on cell proliferation and induction of apoptosis in canine Achilles tendon cells and chondrocytes. Cell growth and proliferation after treating with enrofloxacin for 2–6 days was quantified by a colorimetric 2,3-bis{2-methoxy-4-nitro-5-sulfophenyl}-2H-tetrazolium-5-carboxyanilide inner salt (XTT) assay. The results showed that enrofloxacin could inhibit the proliferation of canine tendon cells and chondrocytes at increasing concentrations (10–200 μg/ml). The inhibition of proliferation of canine tendon cells and chondrocytes after exposure to enrofloxacin were associated with induction of apoptosis, as evidenced by the typical nuclear apoptotic condensed nuclei found using Hoechst 33258 staining. It was demonstrated that canine tendon cells and chondrocytes treated with 200 μg/ml enrofloxacin for 4 days exhibited apoptotic features and fragmentation of DNA. Enrofloxacin also increased the apoptosis of canine tendon cells and chondrocytes in a dose and time-dependent manner. The results indicate that enrofloxacin inhibits cell proliferation, induces apoptosis and DNA fragmentation, which might explain enrofloxacin-induced tendinopathy and cartilage damage.     

Epizootiologic survey for Babesia microti among small wild mammals in northeastern Eurasia and a geographic diversity in the beta-tubulin gene sequences

We previously reported that small wild rodents in Japan harbor two types of novel Babesia microti-like parasites (designated as Hobetsu and Kobe types), but not the type commonly found in the northeastern United States (U.S. type) where human babesiosis is endemic. To determine whether these new types of parasites are distributed in places surrounding Japan, an epizootiologic survey was undertaken in three geographically distant areas in northeastern Eurasia; South Korea, Vladivostok in Russia, and Xinjiang in China. Blood samples were collected from a total of 387 animals comprising 24 species. DNAs extracted from the samples were tested by nested PCR targeting babesial nuclear small-subunit rRNA gene (rDNA), which revealed that small rodents harboring B. microti exist in all three survey areas. Sequence analysis showed that all PCR-positive samples had rDNA sequences virtually identical to that of U.S.-type B. microti. However, when beta-tubulin gene sequences were compared, evident geographic variations were seen. By use of primers specific for each of the beta-tubulin genes of Kobe-, Hobetsu-, and U.S.-type parasites, a type-specific PCR was developed. Parasite with Hobetsu- or Kobe-type sequence was not detected from any of the three survey areas. These findings suggest that U.S.-type B. microti is widely distributed among small wild mammals in temperate zones of not only North America, but also Eurasia, whereas that Hobetsu- and Kobe-type parasites may be uniquely distributed in Japan.     

Isolation and characterization of antimicrobial-resistant Escherichia coli from national horse racetracks and private horse-riding courses in Korea

Limited information is available regarding horse-associated antimicrobial resistant (AR) Escherichia (E.) coli. This study was designed to evaluate the frequency and characterize the pattern of AR E. coli from healthy horse-associated samples. A total of 143 E. coli (4.6%) were isolated from 3,078 samples collected from three national racetracks and 14 private horse-riding courses in Korea. Thirty of the E. coli isolates (21%) showed antimicrobial resistance to at least one antimicrobial agent, and four of the AR E. coli (13.3%) were defined as multi-drug resistance. Most of the AR E. coli harbored AR genes corresponding to their antimicrobial resistance phenotypes. Four of the AR E. coli carried class 1 integrase gene (intI1), a gene associated with multi-drug resistance. Pulsed-field gel electrophoretic analysis showed no genetic relatedness among AR E. coli isolated from different facilities; however, cross-transmissions between horses or horses and environments were detected in two facilities. Although cross-transmission of AR E. coli in horses and their environments was generally low, our study suggests a risk of transmission of AR bacteria between horses and humans. Further studies are needed to evaluate the risk of possible transmission of horse-associated AR bacteria to human communities through horse riders and horse-care workers.     

Immunohistochemical detection of scrapie prion proteins in clinically normal sheep in Pennsylvania.

Following diagnosis of scrapie in a clinically suspect Suffolk sheep, 7 clinically normal flockmates were purchased by the Pennsylvania Department of Agriculture to determine their scrapie status using an immunohistochemical procedure. Two of the 7 euthanized healthy sheep had positive immunohistochemical staining of the prion protein of scrapie (PrP-Sc) in their brains, nictitating membranes, and tonsils. The PrP-Sc was localized in the areas of the brain where, histopathologically, there was neurodegeneration and astrocytosis. The PrP-Sc occurred within germinal centers of the affected nictitating membranes and tonsils and was located in the cytoplasm of the dendrite-like cells, lymphoid cells, and macrophages. These results confirm that immunohistochemical examination of the nictitating membrane can be used as a screen for the presence of scrapie infection in clinically normal sheep at a capable veterinary diagnostic laboratory. In sheep with a PrP-Sc-positive nictitating membrane, the diagnosis of scrapie should be confirmed by histopathology and immunohistochemical examination of the brain following necropsy. Following full validation, immunohistochemistry assays for detection of PrP-Sc in nictitating membrane lymphoid tissues can improve the effectiveness of the scrapie control and eradication program by allowing diagnosis of the disease in sheep before the appearance of clinical signs.     

Composition and mechanism of antitumor effects of Hericium erinaceus mushroom extracts in tumor-bearing mice

We investigated antitumor effects of the following four extracts of freeze-dried Hericium erinaceus mushrooms in Balb/c mice intracutaneously transplanted on the backs with CT-26 colon cancer cells: HWE, hot water extraction by boiling in water for 3 h; MWE, microwaving in 50% ethanol/water at 60 W for 3 min; and ACE and AKE, boiling in 1% HCl or 3% NaOH for 2 h. HWE and MWE with a higher content of β-glucans, determined by an assay kit, than ACE and MKE were active in all bioassays. Gas chromatography/mass spectrometry analyses showed the presence of 40, 27, 16, and 13 compounds, respectively, in the four extracts. Daily intraperitoneal (ip) injections of HWE and MWE for 2 weeks significantly reduced tumor weights by 38 and 41%. Tumor regressions were associated with changes in the following cancer biomarkers as compared to phosphate buffer (PBS)-treated control mice: 2.7- and 2.4-fold increases in cytolytic activity of splenic natural killer (NK) cells; restored nitric oxide production and phagocytosis in peritoneal macrophages to 95-98% of normal levels; ～2-fold increase in released pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6 from macrophages; and ～56 and ～60% reductions in the number of blood vessels inside the tumor. The pro-angiogenic factors vascular endothelial growth factor (VEGF), cyclooxygenase 2 (COX-2), and 5-lipoxygenase (5-LOX) were also significantly reduced in mRNA and protein expression by tumor genes. Enzyme-linked immunosorbent assay of tumor cells confirmed reduced expression of COX-2 and 5-LOX (32 and 31%). Reduced COX-2 and 5-LOX expression down-regulated VEGF expression, resulting in inhibition of neo-angiogenesis inside the tumors. The results indicate that induction of NK activity, activation of macrophages, and inhibition of angiogenesis all contribute to the mechanism of reduction of tumor size.