First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Chard in Taiwan.

Chard (Beta vulgaris var. cicla L.) is a biennial herbaceous plant in the Chenopodiaceae family. It is rich in vitamins and minerals and is one of the most popular traditional vegetables in Taiwan. Chard accessions VI048530 and VI050121 growing in fields at Shanhua, Tainan, showed wilting symptoms in March and April 2013. The initial symptoms of wilt were observed on young green leaves. These symptoms progressed over time to chlorosis, interveinal necrosis, and finally blight. Finally, the plants collapsed and died. Vascular and pith tissues were discolored, especially at the stem base. A whitish mass oozed from the cut end of diseased stems. A total of eight bacterial strains were isolated from stems and roots of wilted chard plants. On tetrazolium chloride (TZC) medium (4), colonies were round to oval, fluidal, and white with a pink or red center after incubation at 30°C for 48 h. A typical hypersensitive reaction was induced within 24 h when the strains were infiltrated into tobacco leaves. Koch's postulates on chard plants were confirmed using the eight strains within a greenhouse, under natural light, with temperature and humidity ranges from 25 to 34°C and 56 to 98%, respectively. Fifteen chard (VI048530) plants at the four- to six-leaf stage were inoculated by soil drenching with 30 ml of a ~108 CFU/ml bacterial suspension. Sterile water was used as negative control. After 4 to 6 days, the first symptoms of wilt were observed on the young chard leaves. The progression of symptom development was identical to that observed in the field. The colony morphology on TZC medium of isolates from the inoculated plants was identical to that previously described from field samples. Pathogenicity of the strains was also tested on tomato (VI005790), eggplant (VI046095), and pepper (PBC1367) plants using the previously described inoculation procedure. The mean disease incidences on tomato, eggplant, and pepper plants were 100% (120/120), 100% (120/120), and 79.2% (95/120) respectively. Latent infection was found in asymptomatic pepper plants (16/120) by a printing method. Polymerase chain reaction (PCR) amplification of total DNA from each strain using the Ralstonia solanacearum-specific primer pair AU759f and AU760r (5) produced the expected 282-bp amplicon. All the isolated strains were identified as biovar 3 based on their capacity to utilize three hexose alcohols (mannitol, sorbitol, and dulcitol) and three disaccharides (lactose, maltose, and cellobiose) (2) to produce acid. Based on the phylotype-specific multiplex PCR assay and the partial egl gene sequence (GenBank accession numbers KM100442 to KM100449) (1), all chard isolates were identified as R. solanacearum phylotype I, sequevar 34. Bacterial wilt symptoms have also been observed on beet (Beta vulgaris L.), a close relative of chard, but beet has not been confirmed as a host plant (3). To our knowledge, this is the first report of chard as a host of R. solanacearum worldwide. References: (1) M. Fegan and P. Prior. Page 449 in: Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. C. Allen et al., eds. APS Press, St. Paul, MN, 2005. (2) A. C. Hayward. J. Appl. Bacteriol. 27:265, 1964. (3) A. Kelman. The Bacterial Wilt caused by Pseudomonas solanacearum. Tech. Bull. No. 99. N.C. Agric. Exp. Stn., 1953. (4) A. Kelman. Phytopathology 44:693, 1954. (5) N. Opina et al. Asia Pac. J. Mol. Biol. Biotechnol. 5:19, 1997.

2-hydroxy-1-oxo-1,2-dihydroisoquinoline-3-carboxylic acid with inbuilt ß-N-hydroxy-γ-keto-acid pharmacophore as HCV NS5B polymerase inhibitors.

The inbuilt 2-N-hydroxy-1-oxo-3-carboxylic acid of isoquinolone was designed as pyrophosphate mimic for hepatitis C NS5B polymerase. Various 2-hydroxy-1-oxo-1,2-dihydroisoquinoline-3-carboxylic acid derivatives 11a-p were synthesized and evaluated as HCV NS5B polymerase inhibitors. Compound 11c exhibited moderate inhibitory potency based on the inorganic pyrophosphate generation (IC50 = 9.5 µM) and based on NTP incorporation by NS5B enzyme (IC50 = 5.9 µM). Compound 11c demonstrated antiviral activity (EC50 = 15.7 µM) and good selectivity in HCV genotype 1b replicon Ava.5 cells. Compound 11c reduced the interaction of NTP to NS5B polymerase. Docking model showed that 11c situated in similar orientation to the bound uridine triphosphate in the active site of NS5B polymerase. As a result, 2-hydroxy-1-oxo-1,2-dihydroisoquinoline-3-carboxylic acid was disclosed as a novel inbuilt ß-N-Hydroxy-γ-keto-acid pharmacophore for HCV NS5B polymerase inhibitors.

Human papillomaviral load changes in low-grade squamous intraepithelial lesions of the uterine cervix.

To better predict risk of progression of low-grade squamous intraepithelial lesions (LSILs) of the uterine cervix in women with human papillomavirus (HPV) infections, 294 baseline cervical specimens from women with LSILs were evaluated. Specimens were tested for HPV DNA using hybrid capture 2 (HC2) and PCR-reverse line blotting. 65 LSILs with HPV DNA types 16, 18, 52, or 58 were examined for physical status, E2/E6 ratio and viral load at two time points, along with patient age. Women with LSILs whose viral loads increased between baseline and 6 month follow-up had a 45% risk of developing HSIL (OR=7.6, 95% CI=1.9-29.4, P<0.01), as evaluated by real-time PCR and a 44% risk (OR=6.1, 95% CI=1.6-22.7, P<0.01), as evaluated by HC2. The two viral load measures correlated well (Person's coefficient, r=0.687, P<0.001). Such evaluations of viral load changes (increased or not increased) through repeat HPV DNA testing could predict progression of disease in LSIL cases of HPV types 16, 18, 52, and 58, which correlates to clinical implications.

The distribution and differential risks of human papillomavirus genotypes in cervical preinvasive lesions: A Taiwan Cooperative Oncologic Group Study.

To clarify the distribution and relative risk of different human papillomavirus (HPV) genotypes in cervical preinvasive lesions, 1246 women with abnormal Papanicolaou smear including atypical squamous cell of unknown significance (ASCUS), atypical glandular cell of unknown significance (AGUS), low-grade squamous intraepithelial lesion (LSIL), and high-grade squamous intraepithelial lesion (HSIL) were enrolled in a multicenter, cross-sectional study. Colposcopy and HPV tests with hybrid capture 2 and polymerase chain reaction-reverse line blot were performed. The prevalences of HPV in ASCUS/AGUS-negative histology, ASCUS/AGUS, LSIL, HSIL, and invasive cancer were 33.8%, 38.3%, 74.9%, 84.3% and 100%, respectively, with an overall positive rate of 68.8%. The most common HPV types were HPV 16 (18.5%), 52 (16.5%), 58 (13.2%), 33, 51, 53, 18, 39, 59, 66, MM8, and 31. In comparing the relative risk of HPV infection in different disease status, LSIL and HSIL/carcinoma had a 4.64 (95% CI: 2.98-7.24) and 10.53 (95% CI: 6.69-16.58) folds of risk of high-risk HPV infection than the negative group. The same was true in mixed HPV infection, but not in low-risk type infection. Looking into each high-risk HPV type, the relative infection risks for LSIL and HSIL/carcinoma, in comparison with the negative group, were 1.67 (0.63-4.43) and 8.67 (3.46-21.70), 2017 (1.01-4.68) and 3.04 (1.42-6.47), and 1.40 (0.52-3.77) and 5.22 (2.07-13.19) for HPV type 16, 52 and 58, respectively. The study confirmed the high prevalence and risky nature of HPV 52 and 58 in Taiwanese population and conveyed the need to include these HPV types in vaccine development.

Factors influencing the switch in the use of antihypertensive medications.

The present study was undertaken to explore factors associated with observance of prescribed antihypertensive medications. Secondary data analysis utilising ambulatory claims of the Taiwan National Health Insurance involved all initially treated hypertensive patients (n=565,048) from June 1, 1997 to June 30, 1998. The main outcome measure was the statistical analysis of factors prompting medication switch and dropout rates. The overall continuity rate was 51%. Specific continuity rates were 40% for calcium antagonists, 36% for angiotensin-converting enzyme inhibitors, 35% for beta-blockers and 29% for diuretics. Less than 30% of patients changed medications and more than 20% of patients changed clinics. The change increased the likelihood of switching medications sevenfold. Switch was minimal for calcium antagonists. Patients taking calcium antagonists were the least likely to switch medications and had the lowest dropout rate. Changing clinics was the most influential medication switch factor.

Characterization of gamma S-crystallin isoforms from lip shark (Chiloscyllium colax): evolutionary comparison between gamma S and beta/gamma crystallins.

gamma S-Crystallin from shark eye lenses, formerly termed beta s crystallin in mammalian lenses, is structurally characterized in this study by cDNA cloning and sequencing. To facilitate sequence characterization of gamma S-crystallin possessing intermediate structural properties between beta- and gamma-crystallins, cDNA mixture was constructed from the poly(A)+ mRNA isolated from shark eye lenses, and amplification by polymerase chain reaction (PCR) was carried out to obtain nucleotide segments encoding multiple shark gamma S-crystallins. Sequencing several positive clones revealed that a multiplicity of isoforms exists in the gamma S-crystallin class of this cartilaginous fish, similar to authentic gamma-crystallin family characterized from the same shark species. Comparison of protein sequences encoded by two representative shark gamma S1 and gamma S2 cDNAs with those published sequences of beta-, gamma-, and gamma S crystallins from bovine, human, bullfrog and carp lenses indicated that there is about 35-64% sequence homology between shark gamma S crystallins and structurally related crystallins from different evolutionary classes, with a higher sequence similarity between shark gamma S and mammalian gamma-crystallins than that of shark gamma S and carp gamma S or bovine gamma S crystallins. A phylogenetic tree constructed on the basis of the sequence divergence among various beta-, gamma-, and gamma S crystallins corroborates the closer relatedness of shark gamma S to authentic gamma-crystallin than to mammalian and teleostean gamma S crystallins. It further strengthens the supposition that ancestral precursors of gamma S-crystallins were present in the shark lens long before the appearance of present-day teleostean and mammalian gamma S-crystallins.

Sequence characterization of gamma-crystallins from lip shark (Chiloscyllium colax): existence of two cDNAs encoding gamma-crystallins of mammalian and teleostean classes.

gamma-Crystallin is a common lens protein of most vertebrate eye lenses and the major protein component in lenses of fishes and in many mammalian species during embryonic and neonatal stages. To facilitate the structural characterization of gamma-crystallin possessing extensive charge heterogeneity, a cDNA mixture was constructed from the poly(A)+ mRNA isolated from shark eye lenses, and amplification by polymerase chain reaction (PCR) was carried out to obtain cDNAs encoding multiple shark gamma-crystallins. Sequencing analysis of multiple positive clones containing PCR-amplified inserts revealed the presence of a multiplicity of isoforms in the gamma-crystallin class of this cartilaginous fish. It was of interest to find that two shark cDNA sequences coexist, one encoding gamma-crystallin (gamma M1) of high methionine content (15.5%) and the other encoding one (gamma M2) of low methionine content (5.1%), each corresponding to the major teleostean and mammalian gamma-crystallins, respectively. Comparison of protein sequences encoded by these two shark cDNAs with published sequences of gamma-crystallins from mouse, bovine, human, frog, and carp lenses indicated that there is about 61-80% sequence homology between different species of the piscine class, whereas only 47-66% is found between mammals and shark. A phylogenetic tree constructed on the basis of sequence divergence among various gamma-crystallin cDNAs revealed the close relatedness between shark gamma M2-crystallin and mammalian gamma-crystallins and that between shark gamma M1 and teleostean gamma-crystallins. The results pointed to the fact that ancestral precursors of gamma-crystallins were present in the sharp lens long before the appearance of modern-day mammalian and teleostean gamma-crystallins.

Inhibition of protein kinase C by snake venom toxins: comparison of enzyme inhibition, lethality and hemolysis among different cardiotoxin isoforms.

Cardiotoxins, neurotoxins and phospholipase A2 (PLA2) are three major classes of toxic components present in the Taiwan cobra, Naja naja atra of the Elapidae family. Cardiotoxins (or called cytotoxins), a group of major polypeptides of around 60 amino-acid residues present abundantly in the elapid family of snakes, comprise about 45-55% of the crude venom of Taiwan cobra. In contrast to another prominent group of structurally similar neurotoxins with well-established acetylcholine receptors and modes of action, cardiotoxins showed no defined cellular targets and very diverse pharmacological functions. A systematic structure/function comparison of these toxins was made by their relative inhibitory effects on protein kinase C (PKC) isolated from mouse brains. Lethality and hemolysis of various cardiotoxin isoforms were also compared in order to shed some insight on the biological targets and mechanisms of these surface-active amphiphilic polypeptides. A structure comparison of these cardiotoxins based on computer model-building revealed that some defined and subtle differences can be detected upon the superposition of these three-dimensional polypeptide chains, which may reflect the intrinsic differences in the hydrophobic peptide segments present on the surface loops of toxin molecules. The differences seem to correlate with different inhibitory activities exhibited by cardiotoxins in contrast to the lack of activity by cobrotoxin and PLA2 on PKC.

In vitro induction of differentiation in HL-60 leukemic cell line by clerodendron fragrans.

The promyelocytic cell line HL-60 could be induced to differentiate into morphologically and functionally mature monocytoid cells (up to 20%) following exposure to the Chinese herb Clerodendron Fragrans (1 mg/ml). This effect was time dependent and appeared to work synergistically with interferon-r in this promotion of differentiation. Our study suggests that Clerodendron Fragrans has potential therapeutic value for the treatment of certain acute myelocytic leukemia putatively caused by a block in the myeloid differentiation process.