Role of S-sulfhydration of RhoA kinase in neuroprotection of hydrogen sulfide against hypoxic injury / 中国药理学通报

Aim To investigate the role of S-sulfhydration of RhoA kinase 2 in the neuroprotection of hydrogen sulfide (H2S) against hypoxic injury. Methods Rat hippocampal neurons were primarily cultured and treated with exogenous H2S donor NaHS (50, 100, 200 (xmol • L"1 ) and S-sulfhydration inhibitor DTT (50 (xmol • L"1 during 4 hours of hypoxia and 12 hours of reoxygenation. Cell viability, the lactate dehydrogenase (LDH) activity and neuron-specific enolase (NSE) activity released from injured neuron to culture supernatant, and the proportion of apoptotic cells were measured to assess the neuroprotection of H2S, and the role of S-sulfhydration in the neuroprotection of H2S was preliminarily explored. In addition, the S-sulf- hydrated proteins in neurons were isolated and purified by modified biotin-switch assay. And then, the RhoA kinase 2 (ROCK2) expression and activity, and S-sul- fhydrated ROCK2 were detected to further confirm the role H2S on the S-sulfhydrated ROCK2 by Western blot and assay kits, respectively. Results The decrease of cell viability, and the increase of LDH and NSE released from injured neuron to culture supernatant and cell apoptosis after hypoxia/ reoxygenation ( H/R) were significantly inhibited by 100 and 200 |imol • L"1 NaHS. Compared with the effect of 200 jimol • L"1 NaHS, the neuroprotection of 200 (xmol • L"1 NaHS could be inhibited by co-application with DTT. Furthermore, 100 and 200 (junol • L"1 NaHS could reduce the expression of R0CK2 protein and restrain ROCK2 activity via promoting the S-sulfhydryl modification of ROCK2 protein in hippocampal neurons. Conclusions H2S exerts protective effect on H/R injury of rat hippocampal neurons via down-regulation of ROCK2 expression and inhibition of R0CK2 activity by S-sulfhydration modification.

Glycyrrhetinic acid binds to the conserved P-loop region and interferes with the interaction of RAS-effector proteins

Members of the proto-oncogene superfamily are indispensable molecular switches that play critical roles in cell proliferation, differentiation, and cell survival. Recent studies have attempted to prevent the interaction of RAS/GTP with RAS guanine nucleotide exchange factors (GEFs), impair RAS-effector interactions, and suppress RAS localization to prevent oncogenic signalling. The present study aimed to investigate the effect of the natural triterpenoic acid inhibitor glycyrrhetinic acid, which is isolated from the roots of plant species, on RAS stability. We found that glycyrrhetinic acid may bind to the P-loop of RAS and alter its stability. Based on our biochemical tests and structural analysis results, glycyrrhetinic acid induced a conformational change in RAS. Meanwhile, glycyrrhetinic acid abolishes the function of RAS by interfering with the effector protein RAF kinase activation and RAS/MAPK signalling.

DTI and DTT in evaluation of effect of childbearing history on female pelvic floor muscles / 实用医学杂志

Objective To investigates the feasibility of diffusion tensor imaging (DTI) and diffusion tensor tracking (DTT) for evaluation of the effect of childbearing history on female pelvic floor muscles. Methods Forty-six healthy females were divided into two groups: nulliparous and primiparous. MR conventional sequences and DTI were acquired. The optimized FA threshold value was obtained by regulating the FA to fiber tracking. The two groups were compared in terms of ADC, FA, VRA and T2-WT. Results (1)The DTT of FA 0.18 got the highest score in fiber tracking . ( 2 ) The ADC of nulliparous subjects and the subjects who had given birth were (1.24 ± 0.11) ×10-3 mm2/s, (1.33 ± 0.11) ×10-3 mm2/s (P = 0.017). There were no statistical differences in FA, VRA and T2-WT between the two groups (P > 0.05). Conclusions The optimized FA threshold of fiber tracking in pelvic floor muscles is 0.18. DTI and DTT may be used to evaluate the effect of childbearing history on female pelvic floor muscles.

A Case of Panagglutination on Antibody Identification in a Multiple Myeloma Patient Receiving Daratumumab / 대한수혈학회지

Herein, we report a patient showing panagglutination in the unexpected antibody identification test after the administration of daratumumab. The patient was a 66-year-old woman who had undergone multiple cycles of chemotherapy and autologous peripheral blood stem cell transplantation for treating multiple myeloma; however, despite treatment, she had relapsed. Therefore, daratumumab, on clinical trials in Korea, started to be administered. After administration of daratumumab, the result of antibody screening test was positive, on the contrary to the result prior to the administration. Moreover, all positive reactions were shown in the antibody identification to the panel cells. After destroying CD38 antigens on the surface of RBCs using DTT, negative results were obtained. Daratumumab—a novel therapeutic human CD38 monoclonal antibody that can be used as targeted immunotherapy—is an FDA-approved drug for treating multiple myeloma. Because CD38 is expressed not only on myeloma cells, but also on red blood cells (RBCs), the use of daratumumab might lead to RBC agglutinations, and thereby resulting in false-positive results on the pre-transfusion tests. Therefore, caution is needed in case of a patient receiving daratumumab. Furthermore, additional test using DTT is required, especially when panagglutination was shown in the antibody identification test, as in this case.

Application of 9. 4 T of DTT in the diagnosis of cerebral gliomas of SD rat / 安徽医科大学学报

Objective To investigate the 9 . 4 T of DTT in the diagnosis of SD rat C6 cerebral gliomas model about the change of CST on tumor progression and pathology confirmed. Methods ①10 μl C6 cell suspension of 106 cells number were implanted into the right caudate nucleus of brain hemisphere of 20 adult male SD rat with ster-eotactic technique. ②Application of 9. 4 T of DTT and DTI, meanwhile the routine T1-weighted imaging(T1WI), T2-weighted imaging(T2WI), contrast-enhanced T1WI,FLAIR imaging and diffusion tensor of the brain were ac-quired. Anatomic relationship between cerebral glioma and surrounding white matter fiber tracts was analyzed and measured the values of FA and MD in different areas on fractional anisotropic ( FA) map, color-coded directional map, three-dimensional (3D) white matter fiber tracts map, then stained with routine HE. Results All of SD rat C6 cerebral gliomas models 3 D white matter fiber tracts map by means of FA maps of DTI were successfully comple-ted. Apparently significant differences of FA values were found in solid tumor, surrounding edema, compared with normal white matter regions ( P0.05);there were significant differences of MD values between solid tumor, surrounding edema and normal white matter region ( P<0.05 ) . Conclusion The 9 . 4 T of DTT offers the optimal visualization of An-atomic relationship between cerebral glioma and surrounding white matter fiber tracts in the different periods of the tumor moder.

The Inhibition of TREK2 Channel by an Oxidizing Agent, 5,5'-dithio- bis (2-nitrobenzoic acid), via Interaction with the C-terminus Distal to the 353rd Amino Acid

TREK (TWIK-RElated K+ channels) and TRAAK (TWIK-Related Arachidonic acid Activated K+ channels) were expressed in COS-7 cells, and the channel activities were recorded from inside-out membrane patches using holding potential of -40 mV in symmetrical 150 mM K+ solution. Intracellular application of an oxidizing agent, 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB), markedly decreased the activity of the TREK2, and the activity was partially reversed by the reducing agent, dithiothreitol (DTT). In order to examine the possibility that the target sites for the oxidizing agents might be located in the C-terminus of TREK2, two chimeras were constructed: TREK2 (1-383)/TASK3C and TREK2 (1-353)/TASK3C. The channel activity in the TREK2 (1-383)/TASK3C chimera was still inhibited by DTNB, but not in the TREK2 (1-353)/TASK3C chimera. These results indicate that TREK2 is inhibited by oxidation, and that the target site for oxidation is located between the amino acid residues 353 and 383 in the C-terminus of the TREK2 protein.

The Effects of DTBNP on Intracellular Ca2+ Signaling in Cultured Bovine Aortic Endothelial Cells

The mechanism underlying oxidant-induced intracellular Ca2+ ([Ca2+]i) increase was studied in cultured bovine aortic endothelial cells (BAECs) using fura-2 AM. In the presence of 2 mM extracellular Ca2+, the application of DTBNP (20microM), a membrane-permeable oxidant, caused an increase in [Ca2+]i, and DTT (2 mM) as a reductant completely reversed the effect of DTBNP. The [Ca2+]i increase induced by DTBNP was also observed in an extracellular Ca2+-free/2 mM EGTA solution, indicating the release of Ca2+ from intracellular store (s). After endoplasmic reticulum was depleted by an IP3-generating agonist, ATP (30microM) or an ER Ca2+ pump inhibitor, thapsigargin (1microM), DTBNP-stressed BAECs showed an increase of [Ca2+]i in Ca2+-free/2 mM EGTA solution. Ratio-differences before and after the application of DTBNP after pretreatment with ATP or thapsigargin were 0.42+/-0.15 and 0.49+/-0.07, respectively (n=7), which are significantly reduced, compared to the control value of 0.72+/-0.07 in a Ca2+-free/2 mM EGTA solution. After the protonophore CCCP (10microM) challenge to release mitochondrial Ca2+, the similar result was obtained. Ratio-difference before and after the application of DTBNP after pretreatment with CCCP was 0.46+/-0.09 (n=7). Simultaneous application of thapsigargin and CCCP completely abolished the DTBNP-induced [Ca2+]i increase. The above results together indicate that the increase of [Ca2+]i by DTBNP resulted from the release of Ca2+ from both endoplasmic reticulum and mitochondria.

Dithothreitol (DTT) treating lymphocytes highly sensitized antibodies in renal pretransplantation patients / 中华器官移植杂志

72 sera of highly sensitized antibody patients were detected by 35 samples of undonor lymphocytes before and after the dithothreitol (DTT) treating.The results showed that 72 sera reacted completely to B lymphocytes in both 4℃ and 37℃,but incompletely to T lymphocytes.After removal of the transplant kidney due to rejection,the sera antibody levels of T or B lymphocytes were higher than those of the patients'sera after blood transfusion or pregnancy.The serum T or B antibody levels of pregnancy were the lowest.In DTT-treated sera of highly sensitized patients,the antibodies were mixed types of IgG and IgM.

DETERMINATION OF REDUCED AND TOTAL VITAMIN C IN BEVERAGE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY / 营养学报

0.05). Conclusion: This method is simple, accurate, feasible for determination of reduced and total VC of beverage.