Journal: Drug Design, Development and Therapy

Article Title: Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action

doi: 10.2147/DDDT.S72765

Figure Lengend Snippet: Midazolam does not attenuate hERG channel surface expression. Effects of midazolam on channel surface expression analyzed by the Western blot technique (upper panel). Image density of the 155 kDa hERG form divided by the 135 kDa hERG form was determined to quantify channel surface expression (lower panel). Incubation with 100 μM As 2 O 3 served as a positive control. Compared with control conditions (lane 2), increasing midazolam concentrations (lanes 3–7) did not result in a significant change of channel surface expression. Abbreviation: hERG, human ether-à-go-go-related gene.

Article Snippet: Midazolam was purchased from Roche (Grenzach, Germany), and stored and handled according to the manufacturer’s specifications.

Techniques: Expressing, Western Blot, Incubation, Positive Control

Journal: Drug Design, Development and Therapy

Article Title: Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action

doi: 10.2147/DDDT.S72765

Figure Lengend Snippet: Concentration dependence of midazolam-induced hERG block. Notes: ( A ) To determine the concentration dependence of midazolam-induced block, a standard voltage protocol was used, eliciting large inward tail currents (see inset). Exemplary current traces elicited by a voltage step to 70 mV prior and after midazolam incubation (100 μM) are displayed. ( B ) Midazolam inhibited hERG channels in a concentration-dependent manner, yielding an IC 50 of 170 μM and a Hill slope of 1.0. Protocol: repetitive pulsing at a frequency of 0.1 Hz, holding potential −80 mV, variable test pulses between −70 mV and +70 mV (400 msec, 10 mV increment), and return pulses to −120 mV (400 msec). ( C ) When analyzed in a mammalian cell line (HEK), the inhibitory effects of midazolam were more pronounced, resulting in a current reduction by 49% and 63% (10 and 30 μM, respectively). A representative current trace under control conditions is shown as an inset. Abbreviation: hERG, human ether-à-go-go-related gene.

Article Snippet: Midazolam was purchased from Roche (Grenzach, Germany), and stored and handled according to the manufacturer’s specifications.

Techniques: Concentration Assay, Blocking Assay, Incubation

Journal: Drug Design, Development and Therapy

Article Title: Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action

doi: 10.2147/DDDT.S72765

Figure Lengend Snippet: Onset and frequency dependence of block. Notes: ( A ) Onset of block was recorded over a period of 30 minutes, using a double-step voltage protocol. Protocol: holding potential −80 mV, first step to +30 mV (400 msec), return pulse −60 mV (400 msec), frequency 0.033 Hz. Peak tail current amplitude was followed to determine the degree of inhibition. Block development was fast, reaching half maximal inhibition at approximately 4 minutes after initiation of midazolam application (n=6). ( B ) To analyze the frequency dependence of block, pulses were applied at frequencies of 1, 0.5, and 0.33 Hz for 60 seconds. Exemplary current traces for the first and last repetitions at a pacing rate of 1 Hz after midazolam incubation as well as the potential protocol are displayed as an inset. Relative current after midazolam incubation is displayed as a function of time. No frequency dependence of block could be observed. Protocol: holding potential −80 mV, first step to +20 mV (300 msec), return pulse −40 mV (300 msec).

Article Snippet: Midazolam was purchased from Roche (Grenzach, Germany), and stored and handled according to the manufacturer’s specifications.

Techniques: Blocking Assay, Inhibition, Incubation

Journal: Drug Design, Development and Therapy

Article Title: Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action

doi: 10.2147/DDDT.S72765

Figure Lengend Snippet: Effects of midazolam on channel inactivation. Notes: ( A ) In order to analyze the effects of midazolam on steady-state inactivation, a double-step voltage protocol was used (see inset). A typical family of current traces is displayed for control conditions ( A ) and after application of 200 μM midazolam ( B ). Current traces were corrected for channel deactivation by extrapolation to the beginning of the second voltage step (see dashed lines and arrows in A ). ( C ) Current-voltage relationship of tail current amplitude. Steady-state inactivation was obtained by dividing the current amplitude by the electrochemical driving force ( D ). Midazolam did not significantly affect the half-maximal inactivation voltage of hERG channels. Protocol: holding potential −80 mV, first test pulse to +40 mV (one second), second test pulse between −140 to +40 mV (20 mV increment, 500 msec). Abbreviation: hERG, human ether-à-go-go-related gene.

Article Snippet: Midazolam was purchased from Roche (Grenzach, Germany), and stored and handled according to the manufacturer’s specifications.

Techniques:

Journal: Drug Design, Development and Therapy

Article Title: Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action

doi: 10.2147/DDDT.S72765

Figure Lengend Snippet: State dependence of midazolam-induced hERG block. Notes: To investigate the state dependence of hERG channel inhibition, two different voltage protocols were chosen. First, oocytes were depolarized from a holding potential of −80 mV using a long pulse to 0 mV (6 seconds). ( A ) Exemplary activating current traces are displayed for control conditions and after incubation with 200 μM midazolam. Fractional block was then calculated by division and plotted versus time ( B ). Within the first seconds, fractional block proceeded in a time-dependent manner, pointing to an open channel block by midazolam. ( C ) Next, a double-step voltage protocol was applied. From a holding potential, channels were activated and inactivated by a long voltage step to +80 mV (3,500 msec). This step was followed by a second step to 0 mV (3,500 msec). Normalized relative current during the second voltage step is displayed in ( D ). In contrast with ( B ), no further development of block could be observed. Abbreviation: hERG, human ether-à-go-go-related gene.

Article Snippet: Midazolam was purchased from Roche (Grenzach, Germany), and stored and handled according to the manufacturer’s specifications.

Techniques: Blocking Assay, Inhibition, Incubation

Journal: Drug Design, Development and Therapy

Article Title: Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action

doi: 10.2147/DDDT.S72765

Figure Lengend Snippet: Midazolam inhibits heterologously expressed hERG channels. Notes: ( A ) A typical family of hERG outward currents elicited by a double-stage voltage protocol. ( B ) Incubation with midazolam resulted in a reduction of hERG currents. ( C ) The corresponding activating current amplitude measured at the end of the first test pulse (first dashed lines in A and B ) as a function of the test pulse potential. ( D ) hERG activation curve: tail current amplitudes (at second dashed lines in A and B ) are shown as a function of the preceding test pulse potential. Midazolam significantly shifted the half-maximal activation voltage of the hERG channels (control −9.17±0.59 mV; midazolam −12.58±0.99 mV). Protocol: holding potential −80 mV, test pulse −70 to +70 mV (2,000 msec) in 10 mV increments, return pulse to −50 mV (2,000 msec). Of note, only every second voltage step is displayed in ( A ) and ( B ) in order to achieve a clearer presentation. Abbreviation: hERG, human ether-à-go-go-related gene.

Article Snippet: Midazolam was purchased from Roche (Grenzach, Germany), and stored and handled according to the manufacturer’s specifications.

Techniques: Incubation, Activation Assay

Journal: Drug Design, Development and Therapy

Article Title: Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action

doi: 10.2147/DDDT.S72765

Figure Lengend Snippet: Mutations of the aromatic pore residues F656A and Y652A affect the inhibitory effects of midazolam. Notes: ( A – C ) Exemplary current traces before and after incubation of midazolam in hERG WT as well as in Y652A hERG and F656A hERG. ( D ) Gives an overview of the observed effects. Compared with hERG WT, the inhibitory effects of midazolam were significantly attenuated in mutant hERG channels. Protocol: holding potential −80 mV, first pulse to +70 mV (400 msec), second pulse to −120 mV (400 msec). Abbreviations: hERG, human ether-à-go-go-related gene; WT, wild-type.

Article Snippet: Midazolam was purchased from Roche (Grenzach, Germany), and stored and handled according to the manufacturer’s specifications.

Techniques: Incubation, Mutagenesis

Journal: PLoS ONE

Article Title: Transcriptional activities of human elongation factor-1α and cytomegalovirus promoter in transgenic dogs generated by somatic cell nuclear transfer

doi: 10.1371/journal.pone.0233784

Figure Lengend Snippet: Analysis of transgenic dogs with EGFP controlled by CMV promoter sequence. (A) Representative image of all born puppies under ultraviolet light exposure, to assess EGFP expression, BTF-963, - 964, -965, -966, -967, and -968, from left to right in order. Claws of three puppies, BTF-965,—967, and -968, showed a detectable EGFP signal, but the signal was undetectable in the other three pupies. (B) PCR analysis to detect whole CMV-EGFP or EGFP construct in genomic DNA of each born dog. GAPDH is the loading control. (C) Detection of GFP signals in fibroblasts isolated from each transgenic dog using fluorescence microscopy and (D) FACS analysis. Scale bars indicate 50 μm. (E) Immunohistochemical analysis showing EGFP expression in brain and muscle tissues from BTF-967 dog. RFP is a negative control of the immunohistochemical reaction.

Article Snippet: GFP expression in primary fibroblasts derived from transgenic pups was analyzed by FACS verse (BD Biosciences).

Techniques: Transgenic Assay, Sequencing, Expressing, Polymerase Chain Reaction, Construct, Isolation, Fluorescence, Microscopy, FACS, Immunohistochemistry, Negative Control

Journal: PLoS ONE

Article Title: Transcriptional activities of human elongation factor-1α and cytomegalovirus promoter in transgenic dogs generated by somatic cell nuclear transfer

doi: 10.1371/journal.pone.0233784

Figure Lengend Snippet: Construction of transgenic canine donor fibroblast with EGFP gene controlled by hEF1α and CMV promoter. (A) Representative images of EGFP-expressing cells at indicated time after transfection with linearized hEF1α- and CMV-EGFP plasmid vectors captured by IncuCyte ® equipment. (B) Quantitative data of green fluorescence object segmentation analysis by the IncuCyte ® basic software. (C) EGFP-positive transgenic donor cells sorted using FACS, 2 weeks after transfection into canine fetal fibroblasts. Scale bars indicate 800 μm. **, P

Article Snippet: GFP expression in primary fibroblasts derived from transgenic pups was analyzed by FACS verse (BD Biosciences).

Techniques: Transgenic Assay, Expressing, Transfection, Plasmid Preparation, Fluorescence, Software, FACS

Journal: PLoS ONE

Article Title: Transcriptional activities of human elongation factor-1α and cytomegalovirus promoter in transgenic dogs generated by somatic cell nuclear transfer

doi: 10.1371/journal.pone.0233784

Figure Lengend Snippet: Analysis of transgenic dogs with EGFP controlled by hEF1α promoter sequence. (A) Representative images showing the four pups produced by SCNT using K9-hEF1α-EGFP fibroblasts. (B) Representative image showing living BTM-881 (right) and BTM-884 (left) dogs without detectable EGFP under ultraviolet light exposure. (C) PCR analysis to detect whole hEF1-EGFP or EGFP construct in genomic DNA of each born dog. GAPDH is the loading control. (D) Detection of EGFP signal in fibroblasts isolated from each transgenic dog using fluorescence images and (E) FACS analysis. (F) Representative images of EGFP-expressing cells treated with 10 μM 5-Aza and/or 0.2 μM TSA for 72 hrs. (G) Quantitative date of cytometry analysis showing the percentage of EGFP-expressing cells in each cell after 5-Aza or/and TSA treatment. (H) Semi-quantitative PCR analysis using isolated mRNA from cells in (F). All scale bars indicate 50 μm. ***, P

Article Snippet: GFP expression in primary fibroblasts derived from transgenic pups was analyzed by FACS verse (BD Biosciences).

Techniques: Transgenic Assay, Sequencing, Produced, Polymerase Chain Reaction, Construct, Isolation, Fluorescence, FACS, Expressing, Cytometry, Real-time Polymerase Chain Reaction

Journal: Experimental Animals

Article Title: Combining isoflurane anesthesia with midazolam and butorphanol in rats

doi: 10.1538/expanim.15-0113

Figure Lengend Snippet: Comparison of MACs in groups treated with isoflurane alone (I) and a combination of midazolam, butorphanol, and isoflurane (MBI). Results are represented as mean ± SD of 7 rats. * P

Article Snippet: One group was administered isoflurane monoanesthesia (Isoflu, DS Pharma Animal Health Co., Ltd., Osaka, Japan), and the other received midazolam (Dormicum, Astellas Pharma Inc., Tokyo, Japan) and butorphanol (Vetorphale, Meiji Seika Pharma Co., Ltd., Tokyo, Japan) followed by isoflurane (MBI).

Techniques: Magnetic Cell Separation

Journal: Experimental Animals

Article Title: Combining isoflurane anesthesia with midazolam and butorphanol in rats

doi: 10.1538/expanim.15-0113

Figure Lengend Snippet: Measured SPO 2 in each anesthetic group. (A): SPO 2 over time in each group. (○): isoflurane-treated group. (●): group treated with a combination of midazolam, butorphanol, and isoflurane. (B): instability in SPO 2 over time represented by coefficient variance (CV). Results are expressed as mean ± SD of 6 rats. *Significant difference between groups ( P

Article Snippet: One group was administered isoflurane monoanesthesia (Isoflu, DS Pharma Animal Health Co., Ltd., Osaka, Japan), and the other received midazolam (Dormicum, Astellas Pharma Inc., Tokyo, Japan) and butorphanol (Vetorphale, Meiji Seika Pharma Co., Ltd., Tokyo, Japan) followed by isoflurane (MBI).

Techniques:

Journal: Experimental Animals

Article Title: Combining isoflurane anesthesia with midazolam and butorphanol in rats

doi: 10.1538/expanim.15-0113

Figure Lengend Snippet: Measured rectal temperature in each anesthetic group. (A): rectal temperature over time in each group. (○): isoflurane-treated group. (●): group treated with a combination of midazolam, butorphanol, and isoflurane. (B): instability in rectal temperature in each group represented as coefficient variance (CV). Results are expressed as mean ± SD in 6 rats. †Significant difference from baseline ( P

Article Snippet: One group was administered isoflurane monoanesthesia (Isoflu, DS Pharma Animal Health Co., Ltd., Osaka, Japan), and the other received midazolam (Dormicum, Astellas Pharma Inc., Tokyo, Japan) and butorphanol (Vetorphale, Meiji Seika Pharma Co., Ltd., Tokyo, Japan) followed by isoflurane (MBI).

Techniques:

Journal: Experimental Animals

Article Title: Combining isoflurane anesthesia with midazolam and butorphanol in rats

doi: 10.1538/expanim.15-0113

Figure Lengend Snippet: Measured heart rate in each anesthetic group. (A): heart rate over time in each group. (○): isoflurane-treated group. (●): group treated with a combination of midazolam, butorphanol, and isoflurane. (B): instability in heart rate in each group represented as coefficient variance (CV). Results are expressed as mean ± SD in 6 rats. †Significant difference from baseline ( P

Article Snippet: One group was administered isoflurane monoanesthesia (Isoflu, DS Pharma Animal Health Co., Ltd., Osaka, Japan), and the other received midazolam (Dormicum, Astellas Pharma Inc., Tokyo, Japan) and butorphanol (Vetorphale, Meiji Seika Pharma Co., Ltd., Tokyo, Japan) followed by isoflurane (MBI).

Techniques:

Journal: Experimental Animals

Article Title: Combining isoflurane anesthesia with midazolam and butorphanol in rats

doi: 10.1538/expanim.15-0113

Figure Lengend Snippet: Measured respiratory rate in each anesthetic group. (A): respiratory rate over time in each group. (○): isoflurane-treated group. (●): group treated with a combination of midazolam, butorphanol, and isoflurane. (B): instability in respiratory rate over time represented by coefficient variance (CV). Results are expressed as mean ± SD in 6 rats. *Significant difference between groups ( P

Article Snippet: One group was administered isoflurane monoanesthesia (Isoflu, DS Pharma Animal Health Co., Ltd., Osaka, Japan), and the other received midazolam (Dormicum, Astellas Pharma Inc., Tokyo, Japan) and butorphanol (Vetorphale, Meiji Seika Pharma Co., Ltd., Tokyo, Japan) followed by isoflurane (MBI).

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: Arterial endothelium creates a permissive niche for expansion of human cord blood hematopoietic stem and progenitor cells

doi: 10.1186/s13287-020-01880-8

Figure Lengend Snippet: Generation of engineered human umbilical arterial endothelial cells by transduction of adenoviral E4orf1 and GFP (HuAECs-E4orf1-GFP). a Schematic illustration of the protocol for test our hypothesis on the role of HuAECs in supporting ex vivo HSPC expansion. b Fluorescence intensity of GFP in HuAECs-E4orf1-GFP and HuVECs-E4orf1-GFP before FACS (above panel); the image of GFP expression in HuAECs-E4orf1-GFP and HuVECs-E4orf1-GFP after FACS (button panel). c Detection of E4ORF1 expression in HuAECs-E4orf1-GFP and HuVECs-E4orf1-GFP by qRT-PCR. d qRT-PCR analysis of arterial and venous markers expression in HuAECs-E4orf1-GFP and HuVECs-E4orf1-GFP. e Comparison of the relative expression of ANGPTL4 , IGF2 , and HOXB4 in HuAECs-E4orf1-GFP and HuVECs-E4orf1-GFP. * P

Article Snippet: Transfected GFP+ cells were enriched via fluorescence-activated cell sorting (FACS) Verse flow cytometer (BD Biosciences, Franklin, NJ, USA).

Techniques: Transduction, Ex Vivo, Fluorescence, FACS, Expressing, Quantitative RT-PCR

Journal: Stem Cell Research & Therapy

Article Title: Arterial endothelium creates a permissive niche for expansion of human cord blood hematopoietic stem and progenitor cells

doi: 10.1186/s13287-020-01880-8

Figure Lengend Snippet: CD34 + hCB cells amplified on HuAECs-E4orf1-GFP exhibited Notch signaling activation. a The CD34 + hCB cell expansion at day 14 and CFU formation in CpE treatment group or with a vehicle control (DMSO) group. b Image of GFP expression in HuAECs-E4orf1-shLV6 (left panel) and HuAECs-E4orf1-shDLL4 (right panel) after FACS. c Expression of DLL4 in HuAECs-E4orf1-shLV6 and HuAECs-E4orf1-shDLL4. d The number of hematopoietic cells and CFU decreased in the HuAECs-E4orf1-shDLL4 group. e Hypothesis for the signal pathways involved in DLL4-mediated promoting proliferation activity of HuAECs-E4orf1 was indicated. NS, no significant difference, * P

Article Snippet: Transfected GFP+ cells were enriched via fluorescence-activated cell sorting (FACS) Verse flow cytometer (BD Biosciences, Franklin, NJ, USA).

Techniques: Amplification, Activation Assay, Expressing, FACS, Activity Assay