Cytotoxic polyketides containing tetramic acid moieties isolated from the fungus Myceliophthora Thermophila: elucidation of the relationship between cytotoxicity and stereoconfiguration.

Five new polyketides that contain tetramic acids, myceliothermophins A-E, were isolated from the thermophilic fungus Myceliophthora thermophila. Two sets of 5-alkyl-5-hydroxyl (or 5-methoxyl)-1H-pyrrol-2(5H)-one diastereomers, myceliothermophins A/B and C/D, were separated as pure compounds by using silica-gel column chromatography and recycling reverse-phase high-performance liquid chromatography (RP-HPLC). The relative configurations of the chiral centers in 5-alkyl-5-hydroxyl (or 5-methoxyl)-1H-pyrrol-2(5H)-one moieties were deduced from NOESY correlations. In the cytotoxic assay, the 5-(2-methylpropyldiene)-1H-pyrrol-2(5H)-one analogue (myceliothermophin E) exhibited inhibition against four cancer cell lines. In addition, the significant inhibitory effect of myceliothermophins A and C and the inactivity of myceliothermophins B and D revealed the importance of the relative configurations of 5-alkyl-5-hydroxyl (or 5-methoxyl)-1H-pyrrol-2(5H)-one moieties on their cytotoxicity potency against cancer cells.

Characterization of a rolling-circle replication plasmid from Thermus aquaticus NTU103.

The thermophilic bacterium Thermus aquaticus NTU103 harbors a 1,965-bp plasmid, pTA103. Sequencing analysis revealed that pTA103 contains two open reading frames. One of the open reading frames (orf2) shares no significant homology with protein in the data bank. The other one has 50% similarity and 34% identity with RepA-like protein of pRm1132f, which is a rolling-circle replication (RCR) plasmid isolated from Sinorhizobium meliloti. S1 nuclease analysis demonstrated that pTA103 contains a single-stranded intermediate, confirming that pTA103 replicates via RCR mechanism. Sequence data also revealed putative double-stranded origin and single-stranded origin sites, indicating the importance of these cis elements in pTA103 replication.

Structural elucidation of phosphoglycolipids from strains of the bacterial thermophiles Thermus and Meiothermus.

The structures of two major phosphoglycolipids from the thermophilic bacteria Thermus oshimai NTU-063, Thermus thermophilus NTU-077, Meiothermus ruber NTU-124, and Meiothermus taiwanensis NTU-220 were determined using spectroscopic and chemical analyses to be 2'-O-(1,2-diacyl-sn-glycero-3-phospho) -3'-O-(alpha-N-acetyl-glucosaminyl)-N-glyceroyl alkylamine [PGL1 (1)] and the novel structure 2'-O-(2-acylalkyldio-1-O-phospho)-3'-O-(alpha-N-acetylglucosaminyl)-N-glyceroyl alkylamine [PGL2 (2)]. PGL2 (2) is the first phosphoglycolipid identified with a 2-acylalkyldio-1-O-phosphate moiety. The fatty acids of the phosphoglycolipids are mainly iso-C(15:0), -C(16:0), and -C(17:0) and anteiso-C(15:0) and -C(17:0). The ratios of PGL2 (2) to PGL1 (1) are significantly altered when grown at different temperatures for three strains, T. thermophilus NTU-077, M. ruber NTU-124, and M. taiwanensis NTU-220, but not for T. oshimai NTU-063. Accordingly, the ratios of iso- to anteiso-branched fatty acids increase when grown at the higher temperature.

Comparison of WHOQOL-bREF and SF-36 in patients with HIV infection.

The purpose of the study was to evaluate the reliability and validity of the two generic instruments, the WHOQOL and the SF-36, for assessing health-related quality of life in 224 patients with HIV infection. The internal consistency ranged from 0.75 to 0.86 across the WHOQOL-BREF domains and from 0.72 to 0.93 across the SF-36 scales. The scores of all WHOQOL-BREF domains and SF-36 scales correlated positively with the measure of happiness, Sat-HRQOL and self-perceived health status, and correlated negatively with the number and intensity of symptoms. Patients with higher CD4 cell counts scored significantly higher on G4 (general health), three WHOQOL-BREF domains, seven SF-36 scales, and PCS (physical component summary). Patients with fewer symptoms and with less intensity of symptoms had significantly higher scores on all four domains of WHOQOL-BREF, eight scales, PCS, and MCS (mental component summary) of the SF-36 scale. The correlations between the physical, psychological, and social domains of the WHOQOL-BREF and PF (physical functioning), MH (mental health), and SF (social functioning) of the SF-36 were 0.51, 0.75, and 0.54, respectively. There is also good correlation between PCS of the SF-36 and the physical domain of the WHOQOL-BREF (r = 0.48), and between MCS and all four domains of the WHOQOL-BREF (r range = 0.60-0.75). The WHOQOL-BREF domains showed fewer floor or ceiling effect than the SF-36 scales. We concluded that both the WHOQOL-BREF and the SF-36 are reliable and valid health related quality-of-life instruments in patients with HIV infection.

Structural determination of the polar glycoglycerolipids from thermophilic bacteria Meiothermus taiwanensis.

The polar glycolipids were isolated from the thermophilic bacteria Meiothermus taiwanensis ATCC BAA-400 by ethanol extraction and purified by Sephadex LH-20 and silica gel column chromatography. The fatty acid composition of O-acyl groups in the glycolipids was obtained by gas chromatography mass spectroscopy analysis on their methyl esters derived from methanolysis and was made mainly of C(15:0) (34.0%) and C(17:0) (42.3%) fatty acids, with the majority as branched fatty acids (over 80%). Removal of O-acyl groups under mild basic conditions provided two glycolipids, which differ only in N-acyl substitution on a hexosamine. Electrospray mass spectroscopy analysis revealed that one has a C(17:0) N-acyl group and the other hydroxy C(17:0) in a ratio of about 1 : 3.5. Furthermore, complete de-lipidation with strong base followed by selective N-acetylation resulted in a homogeneous tetraglycosyl glycerol. The linkages and configurations of the carbohydrate moiety were then elucidated by MS and various NMR analyses. Thus, the major glycolipid from M. taiwanensis ATCC BAA-400 was determined with the following structure: alpha-Galp(1-6)-beta-Galp(1-6)-beta-GalNAcyl(1,2)-alpha-Glc(1,1)-Gro diester, where N-acyl is C(17:0) or hydroxy C(17:0) fatty acid and the glycerol esters were mainly iso- and anteisobranched C(15:0) and C(17:0).

Structure of a major glycolipid from Thermus oshimai NTU-063.

The structure of a major glycolipid isolated from the thermophilic bacteria Thermus oshimai NTU-063 was elucidated. The sugar and fatty acid compositions were determined by GC-MS and HPLC analysis on their methanolysis and methylation derivatives, respectively. After removal of both O- and N-acyl groups by alkaline treatment, the glycolipid was converted to a fully acetylated tetraglycosyl glycerol derivative, the structure of which was then determined by NMR spectroscopy (TOCSY, HSQC, HMBC). Thus, the complete structure of the major glycolipid from T. oshimai NTU-063 was established as beta-Glcp-(1-->6)-beta-Glcp-(1-->6)-beta-GlcpNAcyl-(1-->2)-alpha-Glcp-(1-->1)-glycerol diester. The N-acyl groups on the 2-amino-2-deoxy-glucopyranose residue are C15:0 and C17:0 fatty acids, whereas the fatty acids of glycerol diester are more heterogeneous including both straight and branched fatty acids from C15:0 to C18:0.

Rubrobacter taiwanensis sp. nov., a novel thermophilic, radiation-resistant species isolated from hot springs.

Two novel bacteria, with an optimum growth temperature of approximately 60 degrees C, were isolated from Lu-shan hot springs in the central region of Taiwan. These isolates were aerobic, thermophilic, halotolerant, pink-pigmented, heterotrophic and resistant to gamma-radiation. Both pleomorphic, short, rod-shaped cells and coccoid cells were observed. Strains LS-286 (= ATCC BAA-452 = BCRC 17198) and LS-293T (= ATCC BAA-406T = BCRC 17173T) represented a novel species of the genus Rubrobacter, according to a phylogenetic analysis of the 16S rRNA gene, DNA-DNA hybridization, biochemical features and fatty acid composition. The name Rubrobacter taiwanensis sp. nov. is proposed for this novel species, with LS-293T as the type strain.

Decreased HIV transmission after a policy of providing free access to highly active antiretroviral therapy in Taiwan.

BACKGROUND: Taiwan established a nationwide surveillance system for human immunodeficiency virus (HIV) infection in 1989 and adopted a policy to provide all HIV-infected citizens with free access to highly active antiretroviral therapy (HAART) beginning in April 1997. This provided an opportunity to determine the effect of the widespread use of HAART on the evolution of the HIV epidemic. METHODS: We analyzed national HIV surveillance data. The HIV transmission rate was estimated by use of an exponential model of HIV epidemic evolution, with statistical projection over the interval between infection and detection to fit the surveillance data. RESULTS: By the end of 2002, the cumulative number of HIV-infected citizens in Taiwan had reached 4390 (0.019% of the total population). After free access to HAART was established, the estimated HIV transmission rate decreased by 53% (0.391 vs. 0.184 new cases/prevalent case-year [95% confidence interval, 31%-65%]). There was no statistically significant change in the incidence of syphilis, in the general population or among HIV-positive patients, during the same period. CONCLUSION: Providing free HAART to all HIV-infected citizens was associated with a 53% decrease in the HIV transmission rate and contributed to the control of the HIV epidemic in Taiwan.

Identification of a gene encoding Lon protease from Brevibacillus thermoruber WR-249 and biochemical characterization of its thermostable recombinant enzyme.

A gene encoding thermostable Lon protease from Brevibacillus thermoruber WR-249 was cloned and characterized. The Br. thermoruber Lon gene (Bt-lon) encodes an 88 kDa protein characterized by an N-terminal domain, a central ATPase domain which includes an SSD (sensor- and substrate-discrimination) domain, and a C-terminal protease domain. The Bt-lon is a heat-inducible gene and may be controlled under a putative Bacillus subtilis sigmaA-dependent promoter, but in the absence of CIRCE (controlling inverted repeat of chaperone expression). Bt-lon was expressed in Escherichia coli, and its protein product was purified. The native recombinant Br. thermoruber Lon protease (Bt-Lon) displayed a hexameric structure. The optimal temperature of ATPase activity for Bt-Lon was 70 degrees C, and the optimal temperature of peptidase and DNA-binding activities was 50 degrees C. This implies that the functions of Lon protease in thermophilic bacteria may be switched, depending on temperature, to regulate their physiological needs. The peptidase activity of Bt-Lon increases substantially in the presence of ATP. Furthermore, the substrate specificity of Bt-Lon is different from that of E. coli Lon in using fluorogenic peptides as substrates. Notably, the Bt-Lon protein shows chaperone-like activity by preventing aggregation of denatured insulin B-chain in a dose-dependent and ATP-independent manner. In thermal denaturation experiments, Bt-Lon was found to display an indicator of thermostability value, Tm of 71.5 degrees C. Sequence comparison with mesophilic Lon proteases shows differences in the rigidity, electrostatic interactions, and hydrogen bonding of Bt-Lon relevant to thermostability.

Meiothermus taiwanensis sp. nov., a novel filamentous, thermophilic species isolated in Taiwan.

Two novel filamentous bacterial isolates, strains WR-30T and WR-220, with an optimum growth temperature of approximately 55-60 degrees C were isolated from Wu-rai hot springs in the northern part of Taiwan. These isolates were aerobic, thermophilic, non-sporulating, red-pigmented and heterotrophic and formed extremely long, filamentous trichomes from cells of different lengths. Phylogenetic analysis of 16S rDNA, DNA-DNA hybridization, morphological and biochemical features and fatty acid composition revealed that the isolates represent a novel species of the genus Meiothermus. The name Meiothermus taiwanensis sp. nov. is proposed for this novel species. The type strain of M. taiwanensis is strain WR-30T (= ATCC BAA-399T = CCRC 17170T = DSM 14542T); strain WR-220 (= ATCC BAA400 = CCRC 17171 = DSM 14543) is a reference strain.

Fungi genus and concentration in the air of onion fields and their opportunistic action related to mycotic keratitis.

The authors determined fungi concentrations and genera from onion fields located in monsoon and nonmonsoon areas of Taiwan, as well as concentrations from a county housing location, which served as the comparison site. The authors explored the relationship(s) among onion harvesting, monsoon characteristics, airborne fungal levels, and mycotic eye infection. Airborne fungal levels in the onion field in the nonmonsoon area were the highest, followed by decreasing levels in the monsoon area and control sites, regardless of the sampling periods. The geometric mean of airborne fungal concentrations was the highest during onion harvesting. Cladosporium was the most dominant fungus during pre- and postharvest seasons, whereas Aspergillus, Fusraium, Acremonium, Alternaria, Penicillium, Monilia, Mycelia, and Rhizopus were increased significantly during harvesting. Although fungal concentrations in monsoon onion fields were lower than concentrations measured in the nonmonsoon area, the invasion of dangerous fungal pathogens following eye trauma by airborne sharp exotics (e.g., onion flakes) dispersed in high wind may account for the increase in corneal ulcer cases that occur in the monsoon area.

Pseudoxanthomonas taiwanensis sp. nov., a novel thermophilic, N2O-producing species isolated from hot springs.

Two novel thermophilic bacterial strains, with an optimum growth temperature of between 50 and 60 degrees C, were isolated from the Chi-ban Hot Springs in eastern Taiwan. Strains CB-225 and CB-226(T) were aerobic, thermophilic, non-sporulating, yellow-pigmented heterotrophic organisms. These strains exhibited an unusual denitrification reaction, reducing nitrite, but not nitrate, with the production of N2O only. On the basis of a phylogenetic analysis of 16S rDNA sequences, DNA-DNA similarity data, morphological, physiological and biochemical characteristics, and fatty acid compositions, it was found that the novel strains belonged to the genus Pseudoxanthomonas and represented a novel species within this genus, for which the name Pseudoxanthomonas taiwanensis is proposed; the type strain is CB-226(T) (= ATCC BAA-404(T) = CCRC 17172(T)). P. taiwanensis differs from the only member of the genus Pseudoxanthomonas, the mesophilic species Pseudoxanthomonas broegbernensis, in that it exhibits a higher growth temperature and different morphological characteristics, such as the absence of polar flagella.