[Diagnostic performance of a novel Mycobacterium Tuberculosis specific T-Cell based assay for tuberculosis].

Objective: To evaluate the diagnosic performance of a novel Mycobacterium tuberculosis (MTB) specific T-cell based assay for tuberculosis, which targets the mRNA detection of interferon gamma-induced protein 10 (IP-10). Methods: Suspected tuberculosis patients were prospectively and consecutively recruited in Beijing Chest Hospital between March 2018 and November 2019, and individuals with lower risk of MTB infection were also recruited. IP-10.TB and T-SPOT.TB assays were simulataneously performed on peripheral blood samples. The diagnostic performance of IP-10.TB and T-SPOT.TB were analyzed using the receiver operating characteristic curve. Accordance of IP-10.TB and T-SPOT.TB was analyzed by Cohen's kappa test, while the correlation between the expression level of IP-10 mRNA in IP-10.TB test and the number of SFCs in T-SPOT.TB test were analyzed by Pearson correlation test. Results: A total of 235 patients with tuberculosis, 110 patients with other diseases and 153 individuals with lower risk of MTB infection were included in the final analysis. No significant difference was detected in the rate of indeterminate results between IP-10.TB assay (3/498, 0.60%) and T-SPOT.TB assay (6/498, 1.21%). The total sensitivity and specificity of IP-10.TB assay were 91.3% (95%CI 86.8%-94.6%) and 81.1% (95%CI 75.8%-85.7%). The specificity of IP-10.TB in individuals with lower risk of MTB infection was 98.0% (95%CI 94.4%-99.6%). The total sensitivity and specificity of T-SPOT.TB assay were 93.0% (95%CI 88.9%-96.0%) and 83.8% (95%CI 78.7%-88.1%). The specificity of T-SPOT.TB in individuals with lower risk of MTB infection was 100% (95%CI 97.6%-100.0%). No significant differences were detected in sensitivity and specificity between IP-10.TB and T-SPOT.TB assays (P>0.05). The positive coincidence rate of these 2 methods was 91.0% (95%CI 87.5%-94.5%), and the negative coincidence rate was 88.9% (95%CI 84.9%-92.9%) and the total coincidence rate was 90.0% (95%CI 87.3%-92.6%). The Cohen's kappa value was 0.80 (95%CI 0.75-0.85, P<0.001) between IP-10.TB and T-SPOT.TB assays. Conclusion: These results showed that the diagnostic performance of IP-10.TB was consistent with that in T-SPOT.TB, and this test could be a novel adjunctive tool for the diagnosis of tuberculosis.

Mutational changes of conserved residues in the Q-loop region of transcription factor Rho greatly reduce secondary site RNA-binding.

Transcription factor Rho of Eschericia coli is a ring-shaped homohexameric protein that terminates transcripts by its action on nascent RNAs. To test the functional importance of the phylogenetically highly conserved residues of the Q-loop region, four mutant Rho proteins, S281A, K283A, T286A and D290A, were isolated and analyzed for their biochemical properties. All four proteins were very defective in terminating transcripts in vitro at the bacteriophage lambda tR1 terminator and had corresponding defects in ATP hydrolysis activated by lambda cro RNA. Although the four proteins were normal or near normal in their sensitivity to cleavage with H(2)O(2) in the presence of Fe-EDTA and in their ability to bind to lambda cro RNA and ATP, they were defective in RNA-specific, secondary site interactions. This was indicated by the lack of protection from cleavage at their Q-loops by oligo(C) in the presence of poly(dC), and their defects in ATP hydrolysis activated by oligo(C) in the presence of poly(dC). This evidence, together with the observations that cleavage of the Q-loop residues is protected specifically by RNA, suggests that the Q-loop makes interactions with RNA that are essential for activation of ATP hydrolysis and the termination of transcription.

Oxidative damage to nucleic acids photosensitized by titanium dioxide.

The semiconductor TiO2 is known to have photobiological activity in prokaryotic and eukaryotic cells. Applications of this photobiological activity have been suggested including sterilization of waste water and phototherapy of malignant cells. Here, several model and cellular systems were used to study the mechanism of photocatalysis by TiO2. Treatment of TiO2 (anatase, 0.45 microns), suspended in water containing a spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), with UV radiation (320 nm) resulted in an electron spin resonance (ESR) signal characteristic of the hydroxyl radical. Irradiation of solutions containing calf thymus DNA and TiO2 with UVA (320-400 nm) radiation resulted in hydroxylation of guanine bases. The degree of hydroxylation was dependent on both UVA fluence and amount of TiO2 in suspension. Human skin fibroblasts, preincubated 18 h with 10 micrograms/cm2 TiO2 and then UVA-irradiated (0-58 KJ/m2), showed dose dependent photocytoxicity. RNA, isolated from similarly treated fibroblasts, contained significant levels of photooxidation, measured as hydroxylation of guanine bases. However, no oxidative damage was detectable in cellular DNA. These results suggest that nucleic acids are a potential target for photooxidative damage sensitized by TiO2, and support the view that TiO2 photocatalyzes free radical formation.

In vitro photooxidation of nucleic acids by ultraviolet A radiation.

Although ultraviolet A radiation (UVA, 315-400 nm) has been shown to induce oxidative stress in mammalian cells and skin, the critical chromophore(s) and molecular target(s) involved have not been identified. We examined the role of oxidative damage to nucleic acids induced by UVA. To assess photooxidation of cellular DNA and RNA by UVA, we irradiated human skin fibroblasts with up to 765 kJ/m2 UVA. Cellular DNA and RNA were isolated immediately and enzymatically hydrolyzed to nucleosides. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a primary oxidation product in DNA, and 8-oxo-7,8-dihydroguanosine (8-oxoG), resulting from hydroxylation of guanine in RNA, were measured by HPLC with electrochemical detection. We determined that irradiation of skin fibroblasts with levels of UVA that produced moderate toxicity also resulted in significant levels of guanine hydroxylation in RNA. Lower levels of photooxidation were observed in DNA. These results suggest that measurement of guanine hydroxylation in nucleic acids, particularly in cellular RNA, may be a powerful tool for investigating the photobiological activity of UVA.

Furocoumarin-photosensitized hydroxylation of guanosine in RNA and DNA.

Guanosine hydroxylation was used as a marker for assessing photooxidation of DNA and RNA sensitized by monofunctional and bifunctional furocoumarins. DNA or RNA, treated with sensitizer and UVA light, was enzymatically hydrolyzed, dephosphorylated and then analyzed by reversed-phase HPLC with electrochemical detection. Hydroxylated guanosine, i.e. 8-hydroxy-2'-deoxyguanosine (8-OHdG) or 8-hydroxyguanosine (8-OHG), was quantitated. 3-Carbethoxypsoralen (3-CP) was found to be an efficient photosensitizer for oxidation of guanosine in DNA, resulting in conversion of up to 0.4% of guanosine residues to 8-OHdG. In contrast, dramatically lower levels of guanosine hydroxylation were observed in 3-CP-photosensitized RNA. Psoralen was found to be a more efficient photosensitizer than angelicin in both DNA and RNA. Additional studies of oxidation of 3-CP-photosensitized DNA indicated that double-stranded DNA is 10 times more susceptible to photooxidation than single-stranded DNA, implicating 3-CP binding to DNA as an important mechanistic step in photooxidation of guanosine. The effects of D2O and degassing with argon on photooxidation of guanosine in DNA sensitized by 3-CP were inconsistent with a mechanism involving 1O2. In addition, chelation of adventitious metal ions present in preparations of DNA photosensitized by 3-CP had no effect on hydroxylation of guanosine.

beta-Carotene inhibition of chemically induced toxicity in vivo and in vitro.

In the past several years there has been a great deal of interest in the antioxidant beta-carotene and other micronutrients for their protective potential against various toxic insults. Two studies concerning the protective effects of beta-carotene, which were conducted in our laboratory, are reported here. The first involved the role of beta-carotene in modifying two-stage skin tumorigenesis initiated by 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by phorbol 12-myristate 13-acetate (PMA, TPA). In this study, the protective effects of two types of dietary beta-carotene, a beadlet formulation and crystalline beta-carotene, were compared in two strains of mice (Skh:HR-1 and CR:ORL Sencar). Mice were maintained on food fortified with 3% beta-carotene or on control diets. Mice receiving the beta-carotene-supplemented diets had fewer tumours than mice in the control groups. However, only in the Skh strain of mice was this difference statistically significant. In the second study, an in vitro experiment, BALBc 3T3 mouse fibroblasts were used to determine beta-carotene's accumulation in cells and the ability of these cells to metabolize beta-carotene to vitamin A. This in vitro model was also used to show a beta-carotene protective effect towards 8-MOP phototoxicity. These studies contributed to the increasing evidence of in vivo and in vitro protection by beta-carotene against chemically induced toxicity.

Rapid liquid chromatographic method to distinguish wild salmon from aquacultured salmon fed synthetic astaxanthin.

Analytical methods are needed to determine the presence of color additives in fish. We report a liquid chromatographic (LC) method developed to identify the synthetic form of the color additive astaxanthin in salmon, based on differences in the relative ratios of the configurational isomers of astaxanthin. The distributions of configurational isomers of astaxanthin in the flesh of wild Atlantic and wild Pacific salmon are similar, but significantly different from that in aquacultured salmon. Astaxanthin is extracted from the flesh of salmon, passed through a silica gel Sep-Pak cartridge, and analyzed directly by LC on a Pirkle covalent L-leucine column. No derivatization of the astaxanthin is required-an important advantage of our approach, which is a modification of our previously described method. This method can be used to distinguish between aquacultured and wild salmon. The method has general applicability and can also be used to identify astaxanthins derived from other sources such as Phaffia yeast and Haematococcus pluvialis algae.

Molecular structures and interactions in the yeast kinetochore.

Kinetochores are the elaborate protein assemblies that attach chromosomes to spindle microtubules in mitosis and meiosis. The kinetochores of point-centromere yeast appear to represent an elementary module, which repeats a number of times in kinetochores assembled on regional centromeres. Structural analyses of the discrete protein subcomplexes that make up the budding-yeast kinetochore have begun to reveal principles of kinetochore architecture and to uncover molecular mechanisms underlying functions such as transmission of tension and establishment and maintenance of bipolar attachment. The centromeric DNA is probably wrapped into a compact organization, not only by a conserved, centromeric nucleosome, but also by interactions among various other DNA-bound kinetochore components. The rod-like, heterotetrameric Ndc80 complex, roughly 600 Å long, appears to extend from the DNA-proximal assembly to the plus end of a microtubule, to which one end of the complex is known to bind. Ongoing structural studies will clarify the roles of a number of other well-defined complexes.

Identification of an RNA-binding Site in the ATP binding domain of Escherichia coli Rho by H2O2/Fe-EDTA cleavage protection studies.

Transcription factor Rho is a ring-shaped, homohexameric protein that causes transcript termination through actions on nascent RNAs that are coupled to ATP hydrolysis. The Rho polypeptide has a distinct RNA binding domain of known structure as well as an ATP binding domain for which a structure has been proposed based on homology modeling. Treatment of Rho with H2O2 in the presence of Fe-EDTA caused single-cut cleavage at a number of points that coincide with solvent-exposed loops in both the known and predicted structures, thereby providing support for the validity of the tertiary and quaternary structural models of Rho. The binding of ATP caused one distinct change in the cleavage pattern, a strong protection at a cleavage point in the P-loop of the ATP binding domain. Binding of RNA and single-stranded DNA (poly(dC)) caused strong protection at several accessible parts of the oligosaccharide/oligonucleotide binding (OB) fold in the RNA binding domain. RNA molecules but not DNA molecules also caused a strong, ATP-dependent protection at a cleavage site in the predicted Q-loop of the ATP binding domain. These results suggest that Rho has two distinct binding sites for RNA. Besides the site composed of multiples of the RNA binding domain, to which single-stranded DNA as well as RNA can bind, it has a separate, RNA-specific site on the Q-loop in the ATP binding domain. In the proposed quaternary structure of Rho, the Q-loops from the six subunits form the upper entrance to the hole in the ring-shaped hexamer through which the nascent transcript is translocated by actions coupled to ATP hydrolyses.

beta-Carotene uptake, metabolism, and distribution in BALB/c 3T3 cells.

Although a growing number of epidemiological studies indicate that dietary beta-carotene has anticarcinogenic activity, the mechanism(s) of beta-carotene protection remains to be definitively established. In this context, in vitro studies of beta-carotene have been, and continue to be, valuable. We examined the following critical features in designing an in vitro system for studying the protection action of beta-carotene: 1) form of beta-carotene used for cellular uptake, 2) cellular metabolism of beta-carotene, and 3) subcellular distribution of beta-carotene. It was determined that beta-carotene added to medium in a water-dispersible formulation is readily taken up by BALB/c 3T3 cells and is located predominantly in cellular membranes. Cellular uptake of beta-carotene added to medium in an organic solvent is greatly reduced. It was also found that intracellular retinol increased significantly after a three-day exposure of BALB/c 3T3 cells to media containing beta-carotene. This result suggests that the ability to metabolize beta-carotene to retinoids is not limited to cells of intestinal origin. The results and methodology described here will be useful in the rational design of in vitro assays for elucidating the mechanism(s) of beta-carotene protective effects at the cellular level.

beta-Carotene uptake and effects on intracellular levels of retinol in vitro.

The purpose of the present study was to determine beta-carotene uptake and resultant effects on intracellular levels of retinol in cell lines of varied origin. Human skin fibroblasts, mouse embryonic fibroblasts, rabbit corneal epithelial cells, and rat liver cells were studied. Cells were cultured in medium supplemented with beta-carotene in a water-dispersible beadlet formulation. At selected intervals, cells and media were sampled and analyzed by high-performance liquid chromatography for beta-carotene and retinol content. beta-Carotene was taken up by all four cell lines. An increase in the intracellular levels of retinol was concomitant with beta-carotene uptake in all cell lines. The uptake of beta-carotene and the increase in intracellular retinol were highest in the two fibroblast cell lines. Incubation with media supplemented with crystalline beta-carotene, dissolved in tetrahydrofuran, resulted in significantly lower beta-carotene uptake and intracellular retinol levels. We view these results as a demonstration that a wide variety of cells, cultured in vitro, are able to convert beta-carotene to retinol. Therefore, beta-carotene's provitamin A activity should be carefully considered when the protective effects of beta-carotene in vitro are interpreted.

The protective but nonsynergistic effect of dietary beta-carotene and vitamin E on skin tumorigenesis in Skh mice.

Various epidemiological and experimental studies have indicated that beta-carotene and vitamin E protect against a variety of cancers. This investigation determined whether a synergistic protective effect could be observed against chemically induced skin tumorigenesis in Skh mice by combining these two antioxidants in the diet. Forty-five mice were used in each of four diet groups. Control animals were fed standard mouse chow. Three other groups received the chow supplemented with one of the following: 0.5% beta-carotene, 0.12% vitamin E (added as d-alpha-tocopheryl succinate), or 0.5% beta-carotene + 0.12% vitamin E. Mice were topically treated with a single application of the initiator 7,12-dimethylbenz[a]anthracene and promoted with multiple applications of phorbol 12-myristate 13-acetate. Mice were observed for tumors each week for 27 weeks after initiation. The protective effect of each diet was determined by the decrease in the number of skin tumors in supplemented diet groups compared with that of the control diet group. Decreases in the number of cumulative tumors at Week 27 were 32% for beta-carotene-, 25% for vitamin E-, and 21% for beta-carotene+vitamin E-supplemented diet groups. However, differences in the number of tumors among the three groups supplemented with beta-carotene and/or vitamin E were not statistically significant. Thus, although protection was provided by the individual supplements, there was no synergistic effect for a decrease in the number of chemically induced skin tumors by the simultaneous dietary administration of beta-carotene and vitamin E.