Fluoro-Gold: An alternative viability stain for multicolor flow cytometric analysis.

BACKGROUND: The viability stains propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) are excited at 488 nm, as are the commonly used antibody conjugates fluorescein isothiocyanate (FITC), phycoerythrin (PE), and cyanine 5 dye covalently coupled to R-phycoerythrin (RPE-Cy5). When excited by a single laser, spectral overlap in the emission of PI and 7-AAD with RPE-Cy5 precludes the use of these viability stains for three-color immunophenotyping, particularly when evaluating low levels of marker expression in viable target cells. The ultraviolet excitable dye hydroxystilbamidine methanesulfonate (Fluoro-Gold, or FG) binds to DNA at the A-T-rich regions of the minor groove in permeabilized or dead cells. We assessed the suitability of this dye as a viability stain. METHODS: The ability of FG to detect nonviable cells in fresh and cryopreserved human apheresed peripheral blood cells was compared with that of PI and 7-AAD. The stability of FG staining and the effects of dye and cell concentration on the discrimination of nonviable cells was determined by measuring changes in the median fluorescence of viable and nonviable cells. RESULTS: FG labeling at dye concentrations of 2-8 microM is stable for at least 3 h over a wide range of cell concentrations (4 x 10(5) to 4 x 10(7) cells/ml). Costaining studies and linear regression analysis show that cell viability as determined by FG is strongly correlated with estimates using PI (r = 0.9636) and 7-AAD (r = 0.9879). CONCLUSIONS: FG is a reliable, alternative viability stain that can be used in conjunction with fluorochromes including FITC, PE, and RPE-Cy5 for multicolor analysis using dual-laser instruments.     

用YO-PRO-1和PI联合染色定量检测细胞凋亡

经不同浓度staurosporine处理诱导凋亡的G7细胞样品,分别用YO-PRO-1/PI和AV/PI进行荧光染色,借助流式细胞仪检测凋亡情况,将两种检测方法得到的结果进行统计学分析显示,二者有显著的相关性(r=0.9659,P<0.01),且没有显著性差异(P<0.05);另外,上述凋亡细胞样品经YO-PRO-1/PI染色后在荧光显微镜下计数凋亡细胞比例的结果与AV/PI流式细胞仪的检测结果也有显著的相关性(r=0.9903,P<0.01),且没有显著性差异(P<0.05)。以上这些结果表明,用YO-PRO-1/PI对细胞进行染色、借助流式细胞仪和荧光显微镜均能准确地检测细胞凋亡,可替代AV/PI流式细胞仪方法用于细胞凋亡的检测。     

Susceptibility of irradiated B6D2F1/J mice to Klebsiella pneumoniae administered intratracheally: a pulmonary infection model in an immunocompromised host

Bacteria such as Klebsiella pneumoniae can invade and colonize an immunocompromised host and complicate clinical recovery. In the study reported here, an experimental model of induced pneumonia was developed in 60Co gamma-photon-irradiated mice for the purpose of evaluating efficacy of therapeutic agents. The model was characterized by use of probit analysis of bacterial dose, and microbiologic, and histopathologic results. Bacterial colony-forming-unit (CFU) values producing 50% mortality within 30 days (LD50/30) and their 95% confidence intervals were 4.0 x 10(4) [1.7 x 10(4) - 8.9 x 10(4)] for 0-Gray (Gy)-irradiated mice, 1.9 x 10(4) [7.0 x 10(3) - 4.8 x 10(4)] for 5-Gy-irradiated mice, and 1.0 x 10(3) [2.8 x 10(2) - 3.3 x 10(3)] for 7-Gy-irradiated mice. Probit regression line fits calculated by use of an iterative, weighted least-squares fit, were used to assess a dose-modifying factor (DMF). The DMFs for mortality, compared with that for the 0-Gy dose, with their 95% confidence intervals, were 2.2 [0.63 - 7.7] for the 5-Gy and 38.9 [9.6 -165.0] for 7-Gy doses. The 5-Gy probit line did not significantly differ (P = 0.21) from the 0-Gy probit line (dose ratios did not significantly differ from 1), whereas the 7-Gy probit line differed significantly from the 0-Gy probit line (P < 0.001). These results demonstrate that 7-Gy 60Co gamma-photon radiation in combination with intratracheal K. pneumoniae challenge induces a valid pulmonary infection model in immunocompromised female B6D2F1/J mice.     

Quantification of T-cell-mediated apoptosis in heterogeneous leukemia populations using four-color multiparameter flow cytometry.

BACKGROUND: The unique capacity of dendritic cells to present antigens to naive T cells is being increasingly utilized in cancer therapy. The efficacy of cell-based immunotherapy can be analyzed by determination of cytotoxic activity of T cells toward tumor cells in vitro. This study supplies a flow cytometric method to analyze T-cell-mediated cytotoxic activity toward heterogeneous leukemic cell populations at a single-cell level. METHODS: The fluorescent probe SYTO16 and the dead-cell dye 7-aminoactinomycine-D (7-AAD) were used to identify early and late stages of apoptosis in combination with leukemia cell-identifying markers. Determination of viable, apoptotic, and necrotic cells was performed by inclusion of fluorescent beads. RESULTS: In nine acute myeloid leukemia samples and three leukemic cell lines the use of SYTO16 next to the dead-cell marker 7-AAD significantly increased (P = 0.001) the sensitivity of the cytotoxicity assay as compared with single use of 7-AAD. Analysis of several effector-to-target ratios revealed the ability to determine dose-response effects. Enumeration of absolute numbers resulted in coefficients of variation of 4.1% and 8.4% for cell lines and leukemic samples, respectively. CONCLUSION: The presented flow cytometric cytotoxicity assay enables the study of T-cell-mediated apoptosis in a heterogenous leukemia population.     

Preservation of myocardial function after adenoviral gene transfer in isolated myocardium

Adenoviral gene transfer to the heart represents a promising model for structure-function analyses. Rabbit hearts were subjected to an ex vivo perfusion protocol that achieves gene transfer in >90% of cardiac myocytes. Contractile function of isolated myocardial preparations of these hearts was then observed for 2 days in a recently developed trabecula culture system. In sham-infected hearts, the initial developed force (F(init)) (15.6 +/- 3.7 mN/mm(2); n = 12) did not change significantly after 48 h (17.0 +/- 1.9 mN/mm(2); P = 0.46). In adenovirus-infected preparations, F(init) (14.3 +/- 1. 8 mN/mm(2); n = 21) did not significantly differ from the control (P = 0.75) and was unchanged after 48 h (15.3 +/- 2.5 mN/mm(2); P = 0. 93). After 2 days of continuous contractions, we observed homogenous and high-level expression of the reporter genes LacZ coding for beta-galactosidase and Luc coding for firefly luciferase. Luciferase activity increased more than 2,500-fold from background levels of 8. 7 x 10(3 )+/- 5.0 x 10(3) relative light units (RLU)/mg protein (from hearts transfected with promotorless adenovirus with luciferase transgene construct AdNULLLuc, n = 5) to 23.4 x 10(6)+/- 11.1 x 10(6)RLU/mg protein (from hearts tranfected with adenovirus with Rous sarcoma virus promotor and luciferase transgene construct AdRSVLuc, n = 5) in infected myocardial preparations (P < 0.005). Our results demonstrate a new ex vivo approach to achieve homogenous and high-level expression of recombinant adenoviral genes in contracting myocardium without adverse functional effects.     

新蚜虫疠霉连续液体培养的菌丝生物量、产孢量及杀蚜毒力

继代培养常被疑为虫霉菌种毒力下降或某些生物学性状发生改变的原因之一。从研究新蚜虫疠霉 (Pandoraneoaphidis)固体平板菌落的液体培养获得的初始菌液出发 ,连续 6次继代液培 ,测定了其在萨氏营养液中继代培养生产的菌丝生物量、产孢量及其对桃蚜 (Myzuspersicae)的毒力。在初始菌液的生物量 8 8mg mL和产孢量 7 2× 1 0 5孢子 mg的条件下 ,以三种转接比 (种液 营养液 ,v v)连续 6次继代培养 ,在 1 2 0转接比下的菌丝生物量和产孢量分别为 6 4～ 1 0 0mg mL和 7 3× 1 0 5～ 1 0 8× 1 0 5孢子 mg,在 2 2 0下为 5 7～ 8 5mg mL和 1 0 0× 1 0 5～ 1 2 1× 1 0 5孢子 mg ,在 4 2 0下为 5 5～ 1 0 9mg mL和 6 4× 1 0 5～ 1 0 9× 1 0 5孢子 mg。方差分析表明 ,继代培养对生物量和产孢量均无显著影响 (P <0 0 5)。用初始菌液和 1 2 0转接比下继代培养的菌液制备而成的接种体对桃蚜进行两组毒力测定 ,每一组测定的接种…     

Positron emission tomography with [18F]fluorodeoxyglucose to evaluate neutrophil kinetics during acute lung injury

We measured neutrophil glucose uptake with positron emission tomographic imaging and [18F]fluorodeoxyglucose ([18F]FDG-PET) in anesthetized dogs after intravenous oleic acid-induced acute lung injury (ALI; OA group, n = 6) or after low-dose intravenous endotoxin (known to activate neutrophils without causing lung injury) followed by OA (Etx + OA group, n = 7). The following two other groups were studied as controls: one that received no intervention (n = 5) and a group treated with Etx only (n = 6). PET imaging was performed 1.5 h after initiating experimental interventions. The rate of [3H]deoxyglucose ([3H]DG) uptake was also measured in vitro in cells recovered from bronchoalveolar lavage (BAL) performed after PET imaging. Circulating neutrophil counts fell significantly in animals treated with Etx but not in the other two groups. The rate of [18F]FDG uptake, measured by the influx constant Ki, was significantly elevated (P < 0.05) in both Etx-treated groups (7.9 +/- 2.6 x 10(-3) ml blood x ml lung(-1) x min(-1) in the Etx group, 9.3 +/- 4.8 x 10(-3) ml blood x ml lung(-1) x min(-1) in the Etx + OA group) but not in the group treated only with OA (3.4 +/- 0.8 x 10-3 ml blood x ml lung(-1) x min(-1)) when compared with the normal control (1.6 +/- 0.4 x 10(-3) ml blood x ml lung(-1) x min(-1)). [3H]DG uptake was increased (73 +/- 7%) in BAL neutrophils recovered from the Etx + OA group (P < 0.05) but not in the OA group. Ki and [3H]DG uptake rates were linearly correlated (R2 = 0.65). We conclude that the rate of [18F]FDG uptake in the lungs during ALI reflects the state of neutrophil activation. [18F]FDG-PET imaging can detect pulmonary sequestration of activated neutrophils, despite the absence of alveolar neutrophilia. Thus [18F]FDG-PET imaging may be a useful tool to study neutrophil kinetics during ALI.     

强啡肽抑制离体血管收缩效应的机制

用离体血管电场刺激收缩模型观察到强啡肽明显抑制电场刺激引起的兔耳中心动脉及兔肠系膜上动脉的收缩效应,且呈剂量反应关系,而对股动脉的电场刺激收缩反应无明显影响,强啡肽抑制血管收缩达50％时的用量(IC_(50)值)分别为8.5±1.2×10~(-6)mol/L、5.02±1.3×10~(-7)mol/L及>10~(-6)mol/L。 用药物分析法看到,酚妥拉明(10~(-6)mol/L)可取消电场刺激及去甲肾上腺素引起的血管收缩作用,而强啡肽仅抑制电场刺激致血管收缩作用。 用HPLC法测定孵育液中去甲肾上腺素的含量变化时看到,应用强啡肽(5×10~(-7)mol/L)后孵育液中去甲肾上腺素的含量从对照组的340.56±73.13pg/ml下降至67.91±10.26pg/ml,两组差别有极显著意义(P<0.01)。纳洛酮(10~(-6)mol/L)可完全拮抗强啡肽的这一抑制效应。 以上结果提示强啡肽可能通过抑制交感神经末梢释放去甲肾上腺素,从而产生抑制血管的收缩作用。     

Comparison of multiple assays for kinetic detection of apoptosis in thymocytes exposed to dexamethasone or diethylstilbesterol.

BACKGROUND: Techniques to measure apoptosis are used to study a wide spectrum of conditions, from acquired immune deficiency syndrome (AIDS) to cancer to autoimmune diseases. Therefore, a critical comparison of common assays for apoptosis is warranted. METHODS: The kinetics of apoptosis induction in dexamethasone (DEX)-exposed thymocytes was examined after 2, 4, 8, 12, 26-28, and 48-50 h of culture. An additional aim was to ascertain whether a similar thymic atrophy-inducing hormone, diethylstilbestrol (DES), also directly induces thymocyte apoptosis. Apoptosis was evaluated by flow cytometric examination of cells stained with propidium iodide (PI), 7-aminoactinomycin D (7-AAD), or fluorescein isothiocyante (FITC)-annexin; by forward-and side-scatter (FS, SS) analysis, cell-size analyzer; and through cytopathologic examination. RESULTS: After 4 h of DEX exposure, apoptosis was evident by 7-AAD, annexin, and cytopathological assays, but no cells with sub-diploid DNA content were evident by PI analysis. Maximal apoptosis was evident by all the above flow cytometric techniques at 12 h after DEX exposure. The 7-AAD and annexin assays, which allow discrimination between early apoptosis and late apoptosis/necrosis, were comparable and identified similar apoptotic populations. Appearance of a FSlow/SSincreased population was evident only after 12 h of DEX exposure. Apoptosis could not be detected by any of the above assays in thymocytes exposed to various doses of DES. CONCLUSION: Two of the six assays, 7-AAD and annexin, were similar in detecting apoptosis at an early kinetic time point. Results of both assays were comparable at all time points studied. Our studies imply that DEX and DES induce thymic atrophy, in vivo, by different mechanisms.     

Three methods for producing fertile hemopoietic chimeras in mice

Three methods for producing semiallogeneic (F1----parental) hemopoietic chimeras with retained or regained fertility are detailed here. Prenatal (PN) chimeras were produced by injecting F1 ([BALB/c female x C3H/HeJ male] or [CBA/J female x C57BL/6 male]) fetal liver (days 13-18) or adult bone marrow cells (10(6)-10(7) cells/20 microliters/embryo) into the yolk-sac cavities of days 13-17 gestation BALB/c or CBA/J embryos, respectively, and allowing them to be born naturally. Neonatal (NN) chimeras were made by introducing F1 bone marrow cells (1-2 x 10(7) cells/0.25 ml) into newborn (less than 24 hr old) female mice through the anterior facial vein. Female mice were raised to maturity in both cases. Ovary-transplanted (OT) chimeras were made by first irradiating (9.5 Gy) and repopulating young female adult mice with 10(7) F1 bone marrow cells, followed by bilateral orthotopic transplantation of syngeneic ovarian tissue six weeks later. Females reconstituted with the above three methods were mated with normal syngeneic males and sacrificed at 11-16 days of pregnancy to evaluate hemopoietic chimerism. This was determined in all cases by a radioautographic evaluation of the extent of donor H-2 phenotype marker expression on splenic small lymphocytes, after an indirect labelling of single-cell suspensions with monospecific antibody and [125I]protein-A. Results indicate that hemopoietic chimerism was best in the PN group (0.3-78.1%, mean = 27.1); intermediate in the OT group (5.8-38.2%, mean = 18.1); and low in the NN group (0-14%, with one exception, which was 83.6%). Observed fertility was best for BALB/c host PN chimeras.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leishmania amazonensis: effects of heat shock on ecto-ATPase activity

In this work we demonstrated that promastigotes of Leishmania amazonensis exhibit an Mg-dependent ecto-ATPase activity, which is stimulated by heat shock. The Mg-dependent ATPase activity of cells grown at 22 and 28 degrees C was 41.0+/-5.2 nmol Pi/h x 10(7)cells and 184.2+/-21.0 nmol Pi/h x 10(7)cells, respectively. When both promastigotes were pre-incubated at 37 degrees C for 2h, the ATPase activity of cells grown at 22 degrees C was increased to 136.4+/-10.6 nmol Pi/h x 10(7) whereas that the ATPase activity of cells grown at 28 degrees C was not modified by the heat shock (189.8+/-10.3 nmol Pi/h x 10(7)cells). It was observed that Km of the enzyme from cells grown at 22 degrees C (Km=980.2+/-88.6 microM) was the same to the enzyme from cells grown at 28 degrees C (Km=901.4+/-91.9 microM). In addition, DIDS (4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid) and suramin, two inhibitors of ecto-ATPases, also inhibited similarly the ATPase activities from promastigotes grown at 22 and 28 degrees C. We also observed that cells grown at 22 degrees C exhibit the same ecto-phosphatase and ecto 3'- and 5'-nucleotidase activities than cells grown at 28 degrees C. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat-shock effect on ecto-ATPase activity of cells grown at 22 degrees C were exposed at 37 degrees C for 2h. A comparison between the stimulation of the Mg-dependent ecto-ATPase activity of virulent and avirulent promastigotes by the heat shock showed that avirulent promastigotes had a higher stimulation than virulent promastigotes after heat stress.     

Radiation-induced apoptosis of stem/progenitor cells in human umbilical cord blood is associated with alterations in reactive oxygen and intracellular pH

To investigate the sensitivity of human hematopoietic stem cell populations to radiation and its relevance to intracellular events, specifically alteration in cellular energy production systems, we examined the frequency of apoptotic cells, generation of superoxide anions (O*2-), and changes in cytosol pH in umbilical cord blood (UCB) CD34+/CD38-, CD34+/CD38+ and CD34-/CD38+ cells before and after 5Gy of X-irradiation. Human UCB mononucleated cells were used in this study. After X-irradiation and staining subgroups of the cells with fluorescence (FITC, PE, or CY)-labeled anti-CD34 and anti-CD38 antibodies, analyses were performed by FACScan using as stains 7-amino-actinomycin D (7-AAD) for the detection of apoptosis, and hydroethidine (HE) for the measurement of O*2- generation in the cells. For intracellular pH, image analysis was conducted using confocal laser microscopy after irradiation and staining with carboxy-SNAFR-1. The frequency of apoptotic cells, as determined by cell staining with 7-AAD, was highest in the irradiated CD34+/CD38- cell population, where the level of O*2- detected by the oxidation of HE was also most highly elevated. Intracellular pH measured with carboxy-SNARF-1-AM by image cytometer appeared to be lowest in the same irradiated CD34+/CD38- cell population, and this intracellular pH decreased as early as 4 h post-irradiation, virtually simultaneous with the significant elevation of O*2- generation. These results suggest that the CD34+/CD38- stem cell population is sensitive to radiation-induced apoptosis as well as production of intracellular O*2-, compare to more differentiated CD34+/CD38+ and CD34-/CD38+ cells and that its intracellular pH declines at an early phase in the apoptosis process.     

Myometrial cells induce angiogenesis and salvage damaged myocardium

Characteristically, uterine myometrial cells (MCs) are proliferative, inducing angiogenesis within the female reproductive organ. We evaluated whether MCs implanted into myocardium could also induce angiogenesis and restore heart function after injury. MCs were isolated from the adult rat uterus and cultured for three studies: 1) Intracellular VEGF levels were measured in MCs cultured with progesterone (10(-11), 10(-9), and 10(-7) M) (n = 6 tests per group). 2) Blood vessel density was evaluated 8 days after MCs (3 x 10(6) or 6 x 10(6)), smooth muscle cells (SMCs), or endothelial cells (n = 6 rats per group) were injected with matrigel into the subcutaneous tissue of adult rats. 3) MCs, SMCs (5 x 10(6)/rat), or media were injected into a transmural scar 3 wk after cryoinjury in rat hearts (n = 12 rats per group), and heart function, blood vessel density, and myocardial scar size and thickness were evaluated 5 wk later. In study 1, cultured MCs expressed VEGF, with levels significantly (P < 0.05) upregulated by progesterone at an optimal dose of 10(-11) M. In study 2, MCs injected into the subcutaneous tissue with matrigel induced significantly more blood vessels, especially large-diameter vessels, than did SMCs or endothelial cells (P < 0.01 for all groups). This angiogenic effect was greatest (P < 0.01) at higher doses of MCs and was enhanced by progesterone (10(-11) M). In study 3, MCs implanted into the injured myocardium increased blood vessel density at the implant area, reduced scar size, and improved cardiac function relative to SMCs and media. Overall, MCs induced angiogenesis in vitro and in vivo, prevented cardiac remodeling, and improved heart functional recovery after cardiac injury.     

Lactobacillus rhamnosus strain GG reduces aflatoxin B1 transport, metabolism, and toxicity in Caco-2 Cells

The probiotic Lactobacillus rhamnosus GG is able to bind the potent hepatocarcinogen aflatoxin B1 (AFB1) and thus potentially restrict its rapid absorption from the intestine. In this study we investigated the potential of GG to reduce AFB1 availability in vitro in Caco-2 cells adapted to express cytochrome P-450 (CYP) 3A4, such that both transport and toxicity could be assessed. Caco-2 cells were grown as confluent monolayers on transmembrane filters for 21 days prior to all studies. AFB1 levels in culture medium were measured by high-performance liquid chromatography. In CYP 3A4-induced monolayers, AFB1 transport from the apical to the basolateral chamber was reduced from 11.1%+/-1.9% to 6.4%+/-2.5% (P=0.019) and to 3.3%+/-1.8% (P=0.002) within the first hour in monolayers coincubated with GG (1x10(10) and 5x10(10) CFU/ml, respectively). GG (1x10(10) and 5x10(10) CFU/ml) bound 40.1%+/-8.3% and 61.0%+/-6.0% of added AFB1 after 1 h, respectively. AFB1 caused significant reductions of 30.1% (P=0.01), 49.4% (P=0.004), and 64.4% (P<0.001) in transepithelial resistance after 24, 48, and 72 h, respectively. Coincubation with 1x10(10) CFU/ml GG after 24 h protected against AFB1-induced reductions in transepithelial resistance at both 24 h (P=0.002) and 48 h (P=0.04). DNA fragmentation was apparent in cells treated only with AFB1 cells but not in cells coincubated with either 1x10(10) or 5x10(10) CFU/ml GG. GG reduced AFB1 uptake and protected against both membrane and DNA damage in the Caco-2 model. These data are suggestive of a beneficial role of GG against dietary exposure to aflatoxin.     

Cryopreservation of Specific Pathogen-Free (SPF) Pig Islet Cells: Effect of Culture Time before Cryopreservation and after Thawing

The aim of this study was to determine the optimal conditions (effect of culture time before and after cryopreservation) for cryopreservation of specific pathogen-free pig islet cells. METHODS: (1) Glucose-induced insulin secretion by fresh islet cells cultured for 10 days was compared to that by islet cells cryopreserved 7 days after isolation and cultured 3 days after thawing. (2) Islet cells were cryopreserved 1, 7, or 14 days after isolation and cultured 3, 7, 14, or 21 days after thawing. Islet cell number, insulin content, and insulin response under perifusion tests were investigated. RESULTS: (1) Insulin response by cryopreserved islet cells was identical to that by fresh islet cells (basal/stimulation index: 2. 13 +/- 0.19 vs 2.17 +/- 0.16, n = 4, NS), although the amount of secreted insulin was reduced by 40% (area under the curve: 2136 +/- 198 pM/10(4) cells/180 min vs 3564 +/- 636 pM/10(4) cells/180 min, P = 0.104). (2) Cell number 6 days after thawing was reduced by 54, 40, and 63% when cryopreservations were carried out at D1, D7, and D14. (3) Insulin content in cultured or cryopreserved islet cells increased between 7 and 14 days of culture. (4) Whatever the culture time before and after cryopreservation, insulin secretion in response to glucose was maintained. The insulin release was the highest for islet cells cryopreserved 14 days after isolation and cultured 14 days after thawing (stimulation index: 6.19 +/- 2.68). CONCLUSIONS: SPF pig islet cells remained functional after cryopreservation in polyethylene glycol and it may be important to culture islet cells over 14 days before and after cryopreservation.     

Insulin-like growth factor I (IGF I) induces cortisol production in bovine adrenocortical cells in primary culture

The effects of a physiological dose of IGF I (40 ng/ml approximately 5 x 10(-9) M) on steroidogenesis were studied in bovine adrenal fasciculata cells cultured in serum-free McCoy's medium. They were compared with those of a single dose of ACTH (0.25 ng/ml approximately 10(-10) M) at approximately the concentration inducing half-maximal stimulation. With IGF I, steroidogenesis commenced after 48 h culture and progressively increased throughout the 96-h test period. Expressed as stimulated level/control level ratios, glucocorticoid (cortisol + corticosterone) responses to IGF I after 4 days' culture (2.41 +/- 0.20 (SEM) n = 9) were similar to those obtained with ACTH (2.59 +/- 0.18, n = 9). A combination of the two peptides had a synergistic effect (5.95 +/- 0.79, n = 5). The cortisol/corticosterone ratio increased in the presence of IGF I from 1 +/- 0.19 to 1.76 +/- 0.45 (n = 7, P less than 0.02), although less so than in the presence of ACTH (5.50 +/- 0.98). Moreover, cortisol production was accompanied by androstenedione production (2.36 ng/10(6) cells, n = 3) similar to that induced by ACTH (2.10 ng/10(6) cells, n = 3). These findings together suggest stimulation of 17 alpha-hydroxylase activity. Cell multiplication was unaffected by IGF I. [3H]Thymidine incorporation into DNA reached only 193% +/- 17 (SEM) (n = 4) of control levels, whereas with ACTH it dropped to 60% +/- 5. Our findings show that IGF I alone has no mitogenic effect on adrenocortical cells in vitro, but that it is capable of inducing differentiated steroidogenesis.     

N-methyl-D-glucamine and propidium dyes utilize different permeation pathways at rat P2X(7) receptors

Activation of membrane P2X₇ receptors by extracellular ATP [or its analog 2',3'-O-(4-benzoylbenzoyl)-ATP] results in the opening within several milliseconds of an integral ion channel that is permeable to small cations. If the ATP application is maintained for several seconds, two further sequelae occur: there is a gradual increase in permeability to the larger cation N-methyl-D-glucamine and the cationic propidium dye quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide (YO-PRO-1) enters the cell. The similarity in the time course of these two events has led to the widespread view that N-methyl-D-glucamine and YO-PRO-1 enter through a common permeation pathway, the "dilating" P2X₇ receptor pore. Here we provide two independent lines of evidence against this view. We studied single human embryonic kidney cells expressing rat P2X₇ receptors with patch-clamp recordings of membrane current and with fluorescence measurements of YO-PRO-1 uptake. First, we found that maintained application of the ATP analog did not cause any increase in N-methyl-D-glucamine permeability when the extracellular solution contained its normal sodium concentration, although YO-PRO-1 uptake was readily observed. Second, we deleted a cysteine-rich 18-amino acid segment in the intracellular juxtamembrane region of the P2X₇ receptor. This mutated receptor showed normal YO-PRO-1 uptake but had no permeability to N-methyl-D-glucamine. Together, the clear differential effects of extracellular sodium ions or of mutation of the receptor strongly suggest that N-methyl-D-glucamine and YO-PRO-1 do not enter the cell by the same permeation pathway. ATP; cation channel; permeability; quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide     

A monomer form of the glutathione S-transferase Y7F mutant from Schistosoma japonicum at acidic pH

Dissociation and unfolding of homodimeric glutathione S-transferase Y7F mutant from Schistosoma japonicum (SjGST-Y7F) were investigated at equilibrium using urea as denaturant. The conserved residue Tyr7 plays a central role in the catalytic mechanism and the mutation Tyr-Phe yields an inactive enzyme that is able to bind the substrate GSH with a higher binding constant than the wild type enzyme. Mutant SjGST-Y7F is a dimer at pH 6 or higher and a stable monomer at pH 5 that binds GSH (K value of 1.2x10(5)+/-6.4x10(3)M(-1) at pH 6.5 and 6.3x10(4)+/-1.25x10(3)M(-1) at pH 5). The stability of the SjGST-Y7F mutant was studied by urea induced unfolding techniques (DeltaG(W)=13.86+/-0.63kcalmol(-1) at pH 6.5 and DeltaG(W)=11.22+/-0.25kcalmol(-1) at pH 5) and the monomeric form characterized by means of size exclusion chromatography, fluorescence, and electrophoretic techniques.     

Amperometric detection of perphenazine at a carbon fiber micro-disk bundle electrode by capillary zone electrophoresis

A simple method for determination of perphenazine by capillary zone electrophoresis with amperometric detection is described. The optimum conditions of separation and detection are 1.50 x 10(-3) mol/l Na(2)B(4)O(7)-1.0 x 10(-3) mol/l NaOH (pH 9.9) for the buffer solution, 18 kV for the separation voltage, 5 kV and 5 s for the injection voltage and the injection time, and 0.80 V versus saturated calomel electrode for the detection potential, respectively. The limit of detection is 5.0 x 10(-8) mol/l or 44 amol (S/N=3). The linear range of the calibration curve is 1.00 x 10(-7) to 1.00 x 10(-4) mol/l. The relative standard deviation is 1.5% for the migration time and 2.9% for the electrophoretic current at peak maximum. The method is applied to the determination of perphenazine in human urine.