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1.
Kritsada Pruksaphon Akarin Intaramat Pavinee Simsiriwong Skorn Mongkolsuk Kavi Ratanabanangkoon Joshua D. Nosanchuk Anna Kaltsas Sirida Youngchim 《PLoS neglected tropical diseases》2021,15(5)
Talaromyces marneffei is a thermally dimorphic fungus that causes opportunistic systemic mycoses in patients with AIDS or other immunodeficiency syndromes. The purpose of this study was to develop an immunochromatographic strip test (ICT) based on a solid phase sandwich format immunoassay for the detection of T. marneffei antigens in clinical urine specimens. The T. marneffei yeast phase specific monoclonal antibody 4D1 (MAb4D1) conjugated with colloidal gold nanoparticle was used as a specific signal reporter. Galanthus nivalis Agglutinin (GNA) was adsorbed onto nitrocellulose membrane to serve as the test line. Similarly, a control line was created above the test line by immobilization of rabbit anti-mouse IgG. The immobilized GNA served as capturing molecule and as non-immune mediated anti-terminal mannose of T. marneffei antigenic mannoprotein. The MAb4D1–GNA based ICT showed specific binding activity with yeast phase antigen of T. marneffei, and it did not react with other common pathogenic fungal antigens. The limit of detection of this ICT for T. marneffei antigen spiked in normal urine was approximately 0.6 μg/ml. The diagnostic performance of the ICT was validated using 341 urine samples from patents with culture- confirmed T. marneffei infection and from a control group of healthy individuals and patients with other infections in an endemic area. The ICT exhibited 89.47% sensitivity, 100% specificity, and 97.65% accuracy. Our results demonstrate that the urine-based GNA–MAb4D1 based ICT produces a visual result within 30 minutes and that the test is highly specific for the diagnosis of T. marneffei infection. The findings validate the deployment of the ICT for clinical use. 相似文献
2.
Kurdrid P Subudhi S Cheevadhanarak S Tanticharoen M Hongsthong A 《Molecular biology reports》2007,34(4):261-266
When the gene desD encoding Spirulina Δ6-desaturase was heterologously expressed in E. coli, the enzyme was expressed without the ability to function. However, when this enzyme was co-expressed with an immediate electron
donor, i.e. the cytochrome b
5 domain from Mucor rouxii, the results showed the production of GLA (γ-linolenic acid), the product of the reaction catalyzed by Δ6-desaturase. The results revealed that in E. coli cells, where cytochrome b
5 is absent and ferredoxin, a natural electron donor of Δ6-desaturase, is present at a very low level, the cytochrome b
5 domain can complement for the function of ferredoxin in the host cells. In the present study, the Spirulina-ferredoxin gene was cloned and co-expressed with the Δ6-desaturase in E. coli. In comparison to the co-expression of cytochrome b
5
with the Δ6-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing
the Δ6-desaturase co-expressed with cytochrome b
5 and ferredoxin were exogenously supplied with the intermediate electron donors, NADPH (nicotinamide adenine dinucleotide
phosphate, reduced form) and FADH2 (flavin adenine dinucleotide, reduced form), respectively. The GLA level in these host cells increased drastically, by approximately
50%, compared to the cells without the intermediate electron donors. The data indicated that besides the level of immediate
electron donors, the level of intermediate electron donors is also critical for GLA production. Therefore, if the pools of
the immediate and intermediate electron donors in the cells are manipulated, the GLA production in the heterologous host will
be affected. 相似文献
3.
Wangsakan A McClements DJ Chinachoti P Charles Dickinson L 《Carbohydrate research》2004,339(6):1105-1111
Rotational frame nuclear Overhauser effect spectroscopy (ROESY) and (13)C NMR measurements were carried out to study the molecular interaction between maltodextrin, a digestive byproduct of starch, and an anionic surfactant. Significant differences in chemical shifts were observed when sodium dodecyl sulfate (SDS) was introduced into the maltodextrin (DE 10) solutions. (13)C NMR measurement indicated that there were downfield shifts and broadening of peaks, especially in the region of 75-81 and 100-103 ppm, which were assigned to carbons 1 and 4 of the d-glucopyranose residues of maltodextrin, respectively. ROESY spectra indicated cross-peaks between the SDS and maltodextrin protons. These peaks can arise only in the case of the designated SDS protons and maltodextrin protons being less than 0.5 nm apart for a substantial period of time. The most intense cross-peaks are those between the central CH(2) protons of SDS near 1.2 ppm and the maltodextrin protons ranging from 3.5 to 3.9 ppm. The SDS-H3 CH(2) protons were resolved from the bulk of the SDS protons, with peaks and shoulders at 1.25 ppm, which indicated an especially strong interaction of the SDS hydrophobic tail with MD6 and some less intense interactions with MD2, 4, and 5. 相似文献
4.
Pavinee Kurdrid Jittisak Senachak Matura Sirijuntarut Rayakorn Yutthanasirikul Phuttawadee Phuengcharoen Wattana Jeamton Sittiruk Roytrakul Supapon Cheevadhanarak Apiradee Hongsthong 《Proteome science》2011,9(1):1-17
The present study focused on comparative proteome analyses of low- and high-temperature stresses and potential protein-protein interaction networks, constructed by using a bioinformatics approach, in response to both stress conditions. The data revealed two important points: first, the results indicate that low-temperature stress is tightly linked with oxidative stress as well as photosynthesis; however, no specific mechanism is revealed in the case of the high-temperature stress response. Second, temperature stress was revealed to be linked with nitrogen and ammonia assimilation. Moreover, the data also highlighted the cross-talk of signaling pathways. Some of the detected signaling proteins, e.g., Hik14, Hik26 and Hik28, have potential interactions with differentially expressed proteins identified in both temperature stress conditions. Some differentially expressed proteins found in the Spirulina protein-protein interaction network were also examined for their physical interactions by a yeast two hybrid system (Y2H). The Y2H results obtained in this study suggests that the potential PPI network gives quite reliable potential interactions for Spirulina. Therefore, the bioinformatics approach employed in this study helps in the analysis of phenomena where proteome analyses of knockout mutants have not been carried out to directly examine for specificity or cross-talk of signaling components. 相似文献
5.
Kurdrid P Subudhi S Hongsthong A Ruengjitchatchawalya M Tanticharoen M 《Molecular biology reports》2005,32(4):215-226
Spirulina-acyl-lipid desaturases are membrane-bound enzymes found in thylakoid and plasma membranes. These enzymes carry out the fatty
acid desaturation process of Spirulina to yield γ-linolenic acid (GLA) as the final desaturation product. In this study, Spirulina-Δ6 desaturase encoded by the desD gene was heterologously expressed and characterized in Saccharomyces cerevisiae. We then conducted site-directed mutagenesis of the histidine residues in the three histidine boxes to determine the role
of these amino acid residues in the enzyme function. Our results showed that while four mutants showed complete loss of Δ6-desaturase activity and two mutants showed only trace of the activity, the enzyme activity could be partially restored by
chemical rescue using exogenously provided imidazole. This study reveals that the histidine residues (which have imidazole
as their functional group) in the conserved clusters play a critical role in Δ6-desaturase activity, possibly by providing a di-iron catalytic center. In our previous study, this enzyme was expressed in
Escherichia coli. The results reveal that the enzyme can function only in the presence of an exogenous cofactor, ferredoxin, provided in vitro. This evidence suggests that baker’s yeast has a cofactor that can complement ferredoxin, thought to act as an electron donor
for the Δ6 desaturation in cyanobacteria, including Spirulina. The electron donor of the Spirulina-Δ6 desaturation in yeast is more likely to be cytochrome b5, which is absent in E. coli. This means that the enzyme expressed in S. cerevisiae can catalyze the biosynthesis of the product, GLA, in vivo. 相似文献
6.
Hongsthong A Subudhi S Sirijuntarut M Kurdrid P Cheevadhanarak S Tanticharoen M 《Applied microbiology and biotechnology》2006,72(6):1192-1201
Spirulina-acyl-lipid desaturases are integral membrane proteins found in thylakoid and plasma membranes. These enzymes catalyze the fatty acid desaturation process of Spirulina to yield γ-linolenic acid (GLA) as the final desaturation product. It has been reported that the cyanobacterial desaturases use ferredoxin as an electron donor, whereas the acyl-lipid desaturase in plant cytoplasm and the acyl-CoA desaturase of animals and fungi use cytochrome b
5. The low level of ferredoxin present in Escherichia coli cells leads to an inability to synthesize GLA when the cells are transformed with the Spirulina-∆6 desaturase, desD, and grown in the presence of the reaction substrate, linoleic acid. In this study, Spirulina-∆6 desaturase, encoded by the desD gene, was N-terminally fused and co-expressed with the cytochrome b
5 domain from Mucor rouxii. The product, GLA, made heterologously in E. coli and Saccharomyces cerevisiae, was then detected and analyzed. The results revealed the production of GLA by Spirulina-∆6 desaturase fused or co-expressed with cytochrome b
5 in E. coli cells, in which GLA production by this gene cannot occur in the absence of cytochrome b
5. Moreover, the GLA production ability in the E. coli host cells was lost after the single substitution mutation was introduced to H52 in the HPGG motif of the cytochrome b
5 domain. These results revealed the complementation of the ferredoxin requirement by the fusion or co-expression of the fungal-cytochrome b
5 domain in the desaturation process of Spirulina-∆6 desaturase. Furthermore, the free form of cytochrome b
5 domain can also enhance GLA production by the Spirulina-desD gene in yeast cells. 相似文献
7.
P Kurdrid P Phuengcharoen R Yutthanasirikul S Roytrakul A Paemanee S Cheevadhanarak A Hongsthong 《Biochimie et biologie cellulaire》2012,90(5):621-635
In the present study, electrophoretic mobility shift assays were used to identify temperature responsive elements in the 5' upstream region (5' UTR) of the Spirulina desD gene. Overlapping, synthetic oligonucleotides of both sense and anti-sense strands that spanned the entire 5' UTR of the gene were analyzed. The responsive DNA-binding protein complexes were identified using liquid chromatography-tandem mass spectrometry. The results indicated that the cold-responsive elements were located at -453 to -247, -197 to -151, -105 to -76, and -50 to -1, whereas the low-temperature specific regulatory regions were located at -372 to -352. Moreover, the heat-responsive elements were located at -347 to -243, -197 to -151, and -124 to -1, whereas the high-temperature specific elements were located between -130 to -101 and -30 to -1. In terms of regulatory protein complexes under the two stress conditions, Trx was only detected in the low-temperature responsive protein complex, and divalent cations were essential for the binding of the protein complex to the regulatory elements. Furthermore, Trx was shown to play a critical role as a reducing agent that inactivates the Spirulina desD repressor, GntR. Consequently, the desD gene expression is induced under the low-temperature condition. 相似文献
8.
Kavi Ratanabanangkoon Kae Yi Tan Sukanya Eursakun Choo Hock Tan Pavinee Simsiriwong Teeraporn Pamornsakda Witthawat Wiriyarat Chaiya Klinpayom Nget Hong Tan 《PLoS neglected tropical diseases》2016,10(4)
Snakebite envenomation is a serious medical problem in many tropical developing countries and was considered by WHO as a neglected tropical disease. Antivenom (AV), the rational and most effective treatment modality, is either unaffordable and/or unavailable in many affected countries. Moreover, each AV is specific to only one (monospecific) or a few (polyspecific) snake venoms. This demands that each country to prepare AV against its local snake venoms, which is often not feasible. Preparation of a ‘pan-specific’ AV against many snakes over a wide geographical area in some countries/regions has not been possible. If a ‘pan-specific’ AV effective against a variety of snakes from many countries could be prepared, it could be produced economically in large volume for use in many countries and save many lives. The aim of this study was to produce a pan-specific antiserum effective against major medically important elapids in Asia. The strategy was to use toxin fractions (TFs) of the venoms in place of crude venoms in order to reduce the number of antigens the horses were exposed to. This enabled inclusion of a greater variety of elapid venoms in the immunogen mix, thus exposing the horse immune system to a diverse repertoire of toxin epitopes, and gave rise to antiserum with wide paraspecificity against elapid venoms. Twelve venom samples from six medically important elapid snakes (4 Naja spp. and 2 Bungarus spp.) were collected from 12 regions/countries in Asia. Nine of these 12 venoms were ultra-filtered to remove high molecular weight, non-toxic and highly immunogenic proteins. The remaining 3 venoms were not ultra-filtered due to limited amounts available. The 9 toxin fractions (TFs) together with the 3 crude venoms were emulsified in complete Freund’s adjuvant and used to immunize 3 horses using a low dose, low volume, multisite immunization protocol. The horse antisera were assayed by ELISA and by in vivo lethality neutralization in mice. The findings were: a) The 9 TFs were shown to contain all of the venom toxins but were devoid of high MW proteins. When these TFs, together with the 3 crude venoms, were used as the immunogen, satisfactory ELISA antibody titers against homologous/heterologous venoms were obtained. b) The horse antiserum immunologically reacted with and neutralized the lethal effects of both the homologous and the 16 heterologous Asian/African elapid venoms tested. Thus, the use of TFs in place of crude venoms and the inclusion of a variety of elapid venoms in the immunogen mix resulted in antiserum with wide paraspecificity against elapid venoms from distant geographic areas. The antivenom prepared from this antiserum would be expected to be pan-specific and effective in treating envenomations by most elapids in many Asian countries. Due to economies of scale, the antivenom could be produced inexpensively and save many lives. This simple strategy and procedure could be readily adapted for the production of pan-specific antisera against elapids of other continents. 相似文献
9.
This study involved the fractionation of equine antivenom F(ab′)2 by combined stepwise ammonium sulfate (AS) and caprylic acid (CA) precipitation without intermediate separation of precipitate. Using a microplate format, 55 conditions with combinations of AS (0–20% saturation) and CA (0–5% v/v), were tested. AS significantly reduced the turbidity raised by CA. High specific antibody activity was observed in the area containing 2–5% CA and 10–20% AS. From these results, 12 precipitation conditions were selected for detailed quantitative studies. Two combinations, one with 4% CA and 15% AS and another with 5% CA and 20% AS, gave the highest fold-purification (1.79 and 1.83) with antibody recoveries at 68% and 59%, respectively. These combinations offered a benefit over CA alone in reducing the turbidity and in increasing the purity but not the recovery of antibody. The conditions giving more favorable overall results were with 2% CA alone and another with a combination of 1.5% CA and 10% AS. These preparations of F(ab′)2 were homogeneous and without protein aggregate under size-exclusion HPLC. Lastly, 1 h precipitation showed better results than those of overnight precipitation. These results could be useful for the production of therapeutic antivenoms. 相似文献
10.
Siriwan Rungin Chantra Indananda Pavinee Suttiviriya Worarat Kruasuwan Ratchaniwan Jaemsaeng Arinthip Thamchaipenet 《Antonie van Leeuwenhoek》2012,102(3):463-472
An endophytic Streptomyces sp. GMKU 3100 isolated from roots of a Thai jasmine rice plant (Oryza sativa L. cv. KDML105) showed the highest siderophore production on CAS agar while phosphate solubilization and IAA production were not detected. A mutant of Streptomyces sp. GMKU 3100 deficient in just one of the plant growth promoting traits, siderophore production, was generated by inactivation of a desD-like gene encoding a key enzyme controlling the final step of siderophore biosynthesis. Pot culture experiments revealed that rice and mungbean plants inoculated with the wild type gave the best enhancement of plant growth and significantly increased root and shoot biomass and lengths compared with untreated controls and siderophore-deficient mutant treatments. Application of the wild type in the presence or absence of ferric citrate significantly promoted plant growth of both plants. The siderophore-deficient mutant clearly showed the effect of this important trait involved in plant–microbe interaction in enhancement of growth in rice and mungbean plants supplied with sequestered iron. Our results highlight the value of a substantial understanding of the relationship of the plant growth promoting properties of endophytic actinomycetes to the plants. Endophytic actinomycetes, therefore, can be applied as potentially safe and environmentally friendly biofertilizers in agriculture. 相似文献