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Cost-effective and sensitive aptasensor with guanine chemiluminescence detection capable of simply quantifying thrombin in human serum was developed using thrombin aptamer (TBA), one of the G-quadruplex DNA aptamers, without expensive nanoparticles and complicated procedures. Guanines of G-quadruplex TBA-conjugated carboxyfluorescein (6-FAM) bound with thrombin do not react with 3,4,5-trimethoxylphenylglyoxal (TMPG) in the presence of tetra-n-propylammonium hydroxide (TPA), whereas guanines of free TBA- and TBA-conjugated 6-FAM immobilized on the surface of graphene oxide rapidly react with TMPG to emit light. Thus, guanine chemiluminescence in 5% human serum with thrombin was lower than that without thrombin when TBA-conjugated 6-FAM was added in two samples and incubated for 20 min. In other words, the brightness of guanine chemiluminescence was quenched due to the formation of G-quadruplex TBA-conjugated 6-FAM bound with thrombin in a sample. High-energy intermediate, capable of emitting dim light by itself, formed from the reaction between guanines of TBA and TMPG in the presence of TPA, transfers energy to 6-FAM to emit bright light based on the principle of chemiluminescence energy transfer (CRET). G-quadruplex TBA aptasensor devised using the rapid interaction between TBA-conjugated 6-FAM and thrombin quantified trace levels of thrombin without complicated procedures. The limit of detection (LOD = background + 3 × standard deviation) of G-quadruplex TBA aptasensor with good linear calibration curve, accuracy, precision, and recovery was as low as 12.3 nM in 5% human serum. Using the technology reported in this research, we expect that various types of G-quadruplex DNA aptasensors capable of specifically sensing a target molecule such as ATP, HIV, ochratoxin, potassium ions, and thrombin can be developed.

The gene for LSPI is a lymphocyte-specific gene previously isolated by us using a subtractive hybridization technique. LSPI mRNA is found in normal and transformed B lymphocytes and in normal T lymphocytes but not in transformed T lynrp}iocytes. To study the expression of the mouse LSPI protein, we prepared a polyclonal antiserum spetific for the LSPI protein. Here we report that the gene for LSP1 was expressed in transformed B-lymphoma cell lines and in normal mouse thymocytes as a protein doublet with apparent molecular masses of 52 and 50.5 kilodaltons when analyzed on a sodium dodecyl sulfate-10% polyacrylamide gel. BW5147 cells transfected with an LSP1 cDNA clone expressed only the 52-kilodalton protein. No LSP1 protein was expressed in nine T-lymphoma cell lines tested. Immunofluorescence studies of intact and permeabilized cells and subcellular fractionation experiments showed that the LSPI protein was associated with the cytoplasmic side of the plasma membrane in transformed B-lymphoma cell lines and in normal thymocytes. Using a simple filter-binding assay, we showed that recombinant LSP1 protein was Ca2+ binding, as predicted on the basis of its deduced amino acid sequence. On the basis of the particular expression pattern, the subcellular localization, and the Ca2+-binding property of the LSPI protein, we hypothesize that the LSP1 protein is a lymphocyte-specific component of a signal transduction pathway involved in the regulation of lymphocyte growth.The lymphocyte-specific gene for LSP1 has been isolated by us in the course of a systematic search for cDNA clones which are expressed specifically in all or certain lymphocyte populations but not in nonlymphoid cells (10,12). Such cell type-specific genes are presumably involved in cell typespecific effector functions or, alternatively, they are involved in the regulation of growth or differentiation of the specific cell types in which they are expressed. Northern (RNA) blot analysis has shown that mouse LSP1 mRNA is present in normal B lymphocytes and transformed B-lymphoma cell lines. LSP1 is also expressed in normal thymocytes and in normal functional helper and cytotoxic T-cell lines. However, no LSP1 RNA is present in nine transformed T-lymphoma cell lines tested. The gene for LSP1 is also not expressed in a variety of myeloid cell lines or in nonlymphoid tissues, such as liver, kidney, and heart tissues. A gene with a similar expression pattern can be detected in a series of human cell lines by using the mouse LSP1 cDNA clone as a hybridization probe (12). This lymphocyte-specific expression pattern and the absence of any detectable LSP1 mRNA in transformed T-lymphoma cell lines suggest a role for the LSP1 protein in the regulation of normal lymphocyte growth. Sequence analysis of an LSP1 cDNA clone predicts that the LSP1 protein consists of 330 amino acids with an acidic amino-terminal domain which contains two putative Ca2'-binding sites (12). Given the well-established role of Ca2' as an intracellular mediator of signal transduction (14, 18), ...

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Suspensions of washed, resting‐stage cells of Clostridium bifermentans were incubated with cholic acid under a variety of conditions in an attempt to manipulate the competing 7α‐dehydroxylation and 7α‐dehydrogenation reactions. Important variables thus identified were then tested in batch fermentation. Sodium thioglycollate was observed to inhibit dehydrogenation and to delay dehydroxylation. Zinc ion inhibited both dehydrogenation and dehydroxylation. In the presence of EDTA, near quantitative 7α‐dehydrogenation was achieved with no associated dehydroxylation. Finally, the presence of air in the fermenter headspace enhanced both transformations, possibly through an effect on the redox potential of the medium.

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