Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.108 (TAT)
 2,389 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study was performed to establish optimal nested PCR conditions and a high-yield DNA extraction method for the direct identification of Vibrio vulnificus in clinical specimens. We designed two sets of primers targeting the V. vulnificus hemolysin/cytolysin gene. The target of the first primer set (P1-P2; sense, 5'-GAC-TAT-CGC-ATC-AAC-AAC-CG-3', and antisense, 5'-AGG-TAG-CGA-GTA-TTA-CTG-CC-3', respectively) is a 704-bp DNA fragment. The second set (P3-P4; sense, 5'-GCT-ATT-TCA-CCG-CCG-CTC-AC-3', and antisense, 5'-CCG-CAG-AGC-CGT-AAA-CCG-AA-3', respectively) amplifies an internal 222-bp DNA fragment. We developed a direct DNA extraction method that involved boiling the specimen pellet in a 1 mM EDTA-0.5% Triton X-100 solution. The new DNA extraction method was more sensitive and reproducible than other conventional methods. The DNA extraction method guaranteed sensitivity as well, even when V. vulnificus cells were mixed with other bacteria such as Escherichia coli or Staphylococcus aureus. The nested PCR method could detect as little as 1 fg of chromosomal DNA and single CFU of V. vulnificus. We applied the nested PCR protocol to a total of 39 serum specimens and bulla aspirates from septicemic patients. Seventeen (94.4%) of the 18 V. vulnificus culture-positive specimens were positive by the nested PCR. Eight (42.1%) of the 19 culture-negative samples gave positive nested PCR results.
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PMID:Direct identification of Vibrio vulnificus in clinical specimens by nested PCR. 973 39

Genomic DNA of Blastocystis isolates released into 0.1% Triton X-100 was suitable for amplification and yielded similar results as the genomic DNA extracted with standard kit. The specific B. hominis primers (BH1: GCT TAT CTG GTT GAT CCT GCC AGT and BH2: TGA TCC TTC CGC AGG TTC ACC TAC A) successfully produced the PCR product of about 1,770 bp with all the 7 Blastocystis isolates tested. The restriction fragment length polymorphism (RFLP) patterns yielded by 13 out of 25 restriction endonucleases showed that the 7 isolates could be grouped into 4 subgroups: subgroup-1 consisted of isolate C; subgroup-2 of isolates H4 and H7; subgroup-3 of isolates KP1, Y51 and M12; and subgroup-4 of isolate 27805. The differences between subgroups manifested as clear-cut RFLP patterns. A common band of 230 bp was revealed by Eco R1 in all the Blastocystis isolates tested. The band of about 180 bp was revealed by Alu I, differentiated symptomatic from asymptomatic isolates of this parasite, and might indicate the pathogenicity of this parasite.
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PMID:Restriction enzyme digestion analysis of PCR-amplified DNA of Blastocystis hominis isolates. 1861 39

Scientific interest in microalgal species is growing and, genetic transformation has definitely opened more avenues, in the ongoing research on microphytes. In the present study, we have attempted to transform Chlorella vulgaris by mobilizing double-stranded linear Transfer DNA (T-DNA) comprised of green fluorescent protein (egfp) gene cassette and hygromycin phosphotransferase II (hptII) gene cassette non-covalently bound to TAT peptide, into C. vulgaris cells treated with Triton X-100. The transformed C. vulgaris cells when examined under fluorescent microscope, exhibited green fluorescence in comparison to the untransformed cells. The transformed cells were further screened, and the surviving colonies were sub-cultured, on BG11 medium fortified with Hygromycin. The surviving colonies were confirmed for the presence of integrated T-DNA by Polymerase Chain Reaction with egfp and hptII gene-specific primers. This methodology has potential to substitute the existing tedious transformation methodologies and ease the future studies in microalgae.
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PMID:Genetic transformation of Chlorella vulgaris mediated by HIV-TAT peptide. 3094 86

Unlike the majority of nanomaterials designed for cellular uptake via endocytic pathways, some of the functional nanoparticles and nanospheres directly enter the cytoplasm without overt biomembrane injuries. Previously, we have shown that a water-soluble nanoaggregate composed of amphiphilic random copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and n-butyl methacrylate (BMA), poly(MPC- random-BMA) (PMB), passes live cell membranes in an endocytosis-free manner. Yet, details in its translocation mechanism remain elusive due to the lack of proper analytical methods. To understand this phenomenon experimentally, we elaborated the original pH perturbation assay that is extremely sensitive to the pore formation on cell membranes. The ultimate sensitivity originates from the detection of the smallest indicator H+ (H3O+) passed through the molecularly sized transmembrane pores upon challenge by exogenous reagents. We revealed that water-soluble PMB at the 30 mol % MPC unit (i.e., PMB30W) penetrated into the cytosol of model mammalian cells without any proton leaks, in contrast to conventional cell-penetrating peptides, TAT and R8 as well as the surfactant, Triton X-100. While exposure of PMB30W permeabilized cytoplasmic lactate dehydrogenase out of the cells, indicating the alteration of cell membrane polarity by partitioning of amphiphilic PMB30W into the lipid bilayers. Nevertheless, the biomembrane alterations by PMB30W did not exhibit cytotoxicity. In summary, elucidating translocation mechanisms by proton dynamics will guide the design of nanomaterials with controlled permeabilization to cell membranes for bioengineering applications.
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PMID:Translocation Mechanisms of Cell-Penetrating Polymers Identified by Induced Proton Dynamics. 3109 2