Characteristics of membrane currents evoked by photoreleased inositol trisphosphate in Xenopus oocytes

1992 ◽  
Vol 263 (1) ◽  
pp. C154-C165 ◽  
Author(s):  
I. Parker ◽  
I. Ivorra
Keyword(s):  
Xenopus Oocytes ◽  
Threshold Level ◽  
Dose Dependence ◽  
Evoked Responses ◽  
Flash Duration ◽  
Competitive Antagonist ◽  
Study Characteristics ◽  
Time Required ◽  
Threshold Duration ◽  
Interflash Interval

Photorelease of inositol 1,4,5-trisphosphate (InsP3) from a caged precursor was used to study characteristics of Ca(2+)-activated Cl- currents activated in Xenopus oocytes by the InsP3-Ca2+ signaling pathway. Photolysis flashes shorter than a threshold duration evoked no response, but the current amplitude then grew about linearly as the flash duration was further lengthened. Currents directly evoked by photorelease of Ca2+ from a caged precursor grew linearly with increasing flash duration and showed a small threshold before they were activated. However, the major part of the threshold of InsP3-evoked responses appears to arise because a certain concentration of InsP3 (estimated to be approximately 60 nM) is required to evoke Ca2+ liberation. Subthreshold conditioning flashes potentiated responses to subsequent flashes, and the potentiation increased linearly with increasing conditioning flash duration before abruptly declining. The potentiation decayed exponentially with a time constant of approximately 17 s with increasing interflash interval. Currents evoked by photoreleased InsP3 began after a latency that shortened from 10 s or longer to 100 ms as the photolysis intensity was increased. This dose dependence of the latency could be quantitatively explained by the time required for the InsP3 concentration to rise above threshold. Intracellular injection of heparin (a competitive antagonist at the InsP3 receptor) increased the threshold for InsP3 action, as did increased temperature. We conclude that several characteristics of InsP3-evoked responses, including their dose dependence, latency, and facilitation with paired stimuli, arise because a distinct threshold level of InsP3 is required to evoke release of Ca2+ from intracellular stores.

scholarly journals Transcription elongation in the human c-myc gene is governed by overall transcription initiation levels in Xenopus oocytes.

1993 ◽  
Vol 13 (2) ◽  
pp. 1296-1305 ◽  
Author(s):  
C A Spencer ◽  
M A Kilvert
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Incubation Time ◽  
Transcription Initiation ◽  
Xenopus Oocytes ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Threshold Level ◽  
Specific Effects ◽  
Myc Gene

Both transcription initiation and transcription elongation contribute to the regulation of steady-state c-myc RNA levels. We have used the Xenopus oocyte transcription assay to study premature transcription termination which occurs in the first exon and intron of the human c-myc gene. Previous studies showed that after injection into Xenopus oocytes transcription from the c-myc P1 promoter resulted in read-through transcripts whereas transcription from the stronger P2 promoter resulted in a combination of prematurely terminated and read-through transcripts. We now demonstrate that this promoter-specific processivity results from the overall amount of RNA polymerase II transcription occurring from either promoter. Parameters that reduce the amount of transcription from P1 or P2, such as decreased concentration of template injected or decreased incubation time, result in a reduction in the ratio of terminated to read-through c-myc transcripts. Conversely, when transcription levels are increased by higher concentrations of injected template, increased incubation time, or coinjection with competing template, the ratio of terminated to read-through transcripts increases. We hypothesize that an RNA polymerase II processivity function is depleted above a threshold level of transcription initiation, resulting in high levels of premature transcription termination. These findings account for the promoter-specific effects on transcription elongation previously seen in this assay system and suggest a mechanism whereby limiting transcription elongation factors may contribute to transcription regulation in other eukaryotic cells.

Dose-Dependent “Activation Energy” For Blistering Phenomenon In Hydrogen-Implanted Silicon

1998 ◽  
Vol 513 ◽  
Author(s):  
S. W. Bedell ◽  
W. A. Lanford
Keyword(s):  
Activation Energy ◽  
Annealing Temperature ◽  
Arrhenius Plot ◽  
Dose Dependence ◽  
Qualitative Explanation ◽  
Hydrogen Distribution ◽  
Measured Activation ◽  
Time Required ◽  
Dose Dependent ◽  
Microscope Stage

ABSTRACTSilicon samples were implanted with 100 keV hydrogen at doses ranging from 4.9 to 9.9 × 1016 H/cm2. The samples were then annealed in an argon atmosphere using a special furnace mounted on a microscope stage. The time required to observe the onset of Si blistering was then measured as a function of temperature for the various hydrogen doses. An Arrhenius plot of the time-temperature data was made and an “activation energy” was calculated for each dose. The measured activation energies range from 2.6 to 1.1 eV corresponding to 4.9 to 9.9 × 1016 H/cm2, respectively. A qualitative explanation of the dose dependence is given in terms of a stressinduced lowering of the Si-Si bond energy. The hydrogen distribution as a function of annealing temperature was monitored using the 1H(15N,αγ)12C nuclear reaction. The results from the hydrogen profiling indicate that the hydrogen profile flattens at 500°C and concentrates at 550 °C.

scholarly journals Functional consequences of lidocaine binding to slow-inactivated sodium channels.

1996 ◽  
Vol 107 (5) ◽  
pp. 643-658 ◽  
Author(s):  
J R Balser ◽  
H B Nuss ◽  
D N Romashko ◽  
E Marban ◽  
G F Tomaselli
Keyword(s):  
Xenopus Oocytes ◽  
Potential Contribution ◽  
Channel Block ◽  
Na Channels ◽  
Alpha Subunits ◽  
Pathological Conditions ◽  
Functional Consequences ◽  
Delayed Recovery ◽  
Time Required ◽  
Block Of Na Channels

Na channels open upon depolarization but then enter inactivated states from which they cannot readily reopen. After brief depolarizations, native channels enter a fast-inactivated state from which recovery at hyperpolarized potentials is rapid (< 20 ms). Prolonged depolarization induces a slow-inactivated state that requires much longer periods for recovery (> 1 s). The slow-inactivated state therefore assumes particular importance in pathological conditions, such as ischemia, in which tissues are depolarized for prolonged periods. While use-dependent block of Na channels by local anesthetics has been explained on the basis of delayed recovery of fast-inactivated Na channels, the potential contribution of slow-inactivated channels has been ignored. The principal (alpha) subunits from skeletal muscle or brain Na channels display anomalous gating behavior when expressed in Xenopus oocytes, with a high percentage entering slow-inactivated states after brief depolarizations. This enhanced slow inactivation is eliminated by coexpressing the alpha subunit with the subsidiary beta 1 subunit. We compared the lidocaine sensitivity of alpha subunits expressed in the presence and absence of the beta 1 subunit to determine the relative contributions of fast-inactivated and slow-inactivated channel block. Coexpression of beta 1 inhibited the use-dependent accumulation of lidocaine block during repetitive (1-Hz) depolarizations from -100 to -20 mV. Therefore, the time required for recovery from inactivated channel block was measured at -100 mV. Fast-inactivated (alpha + beta 1) channels were mostly unblocked within 1 s of repolarization; however, slow-inactivated (alpha alone) channels remained blocked for much longer repriming intervals (> 5 s). The affinity of the slow-inactivated state for lidocaine was estimated to be 15-25 microM, versus 24 microM for the fast-inactivated state. We conclude that slow-inactivated Na channels are blocked by lidocaine with an affinity comparable to that of fast-inactivated channels. A prominent functional consequence is potentiation of use-dependent block through a delay in repriming of lidocaine-bound slow-inactivated channels.

scholarly journals Inhibition of topoisomerase II does not inhibit transcription of RNA polymerase I and II genes.

1990 ◽  
Vol 10 (6) ◽  
pp. 2893-2900 ◽  
Author(s):  
M Dunaway
Keyword(s):  
Rna Polymerase ◽  
Thymidine Kinase ◽  
Topoisomerase Ii ◽  
Xenopus Oocytes ◽  
Initial Period ◽  
Rna Polymerase I ◽  
Time Required ◽  
Polymerase I ◽  
Linear Product

Injection of VM-26 (teniposide) into Xenopus oocytes inhibits the activity of topoisomerase II but does not inhibit transcription by RNA polymerases I and II. A specific assay for topoisomerase II, resolution of catenated DNA molecules into product rings, was used to quantitate VM-26 inhibition in vivo. When catenanes were injected without VM-26, about 60% of them were separated into product rings in the first 5 min after injection, and decatenation of the remainder was complete within 15 min. When VM-26 was coinjected, 60% of the catenanes were separated into product rings in the first 5 min after injection, but the remaining 40% were stable over the next 40 min. At 1 h after injection catenanes were no longer detected in the gel analysis, but the increasing numbers of linear product rings indicated that topoisomerase II continued to be inhibited by VM-26. These results suggest that a short lag of approximately 5 min is required for VM-26 to inhibit topoisomerase II and that after this initial period topoisomerase II is inhibited by more than 90%. There was no detectable decrease in transcription of injected rRNA and thymidine kinase (TK) genes or in the activity of the rRNA enhancer when these transcription templates were coinjected with VM-26. The time required for assembly of injected DNA into chromatin doubled in the presence of VM-26.

scholarly journals Systems approach to assessing and improving local human research Institutional Review Board performance

2018 ◽  
Vol 2 (2) ◽  
pp. 103-109 ◽  
Author(s):  
John Fontanesi ◽  
Anthony Magit ◽  
Jennifer J. Ford ◽  
Han Nguyen ◽  
Gary S. Firestein
Keyword(s):  
Systems Approach ◽  
Predictive Analytics ◽  
Institutional Review Boards ◽  
Quality Improvement Effort ◽  
Human Research ◽  
Predictive Algorithm ◽  
Institutional Review ◽  
Coverage Analysis ◽  
Study Characteristics ◽  
Time Required

ObjectiveTo quantifying the interdependency within the regulatory environment governing human subject research, including Institutional Review Boards (IRBs), federally mandated Medicare coverage analysis and contract negotiations.MethodsOver 8000 IRB, coverage analysis and contract applications initiated between 2013 and 2016 were analyzed using traditional and machine learning analytics for a quality improvement effort to improve the time required to authorize the start of human research studies.ResultsStaffing ratios, study characteristics such as the number of arms, source of funding and number and type of ancillary reviews significantly influenced the timelines. Using key variables, a predictive algorithm identified outliers for a workflow distinct from the standard process. Improved communication between regulatory units, integration of common functions, and education outreach improved the regulatory approval process.ConclusionsUnderstanding and improving the interdependencies between IRB, coverage analysis and contract negotiation offices requires a systems approach and might benefit from predictive analytics.

Transcription elongation in the human c-myc gene is governed by overall transcription initiation levels in Xenopus oocytes

1993 ◽  
Vol 13 (2) ◽  
pp. 1296-1305
Author(s):  
C A Spencer ◽  
M A Kilvert
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Incubation Time ◽  
Transcription Initiation ◽  
Xenopus Oocytes ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Threshold Level ◽  
Specific Effects ◽  
Myc Gene

Both transcription initiation and transcription elongation contribute to the regulation of steady-state c-myc RNA levels. We have used the Xenopus oocyte transcription assay to study premature transcription termination which occurs in the first exon and intron of the human c-myc gene. Previous studies showed that after injection into Xenopus oocytes transcription from the c-myc P1 promoter resulted in read-through transcripts whereas transcription from the stronger P2 promoter resulted in a combination of prematurely terminated and read-through transcripts. We now demonstrate that this promoter-specific processivity results from the overall amount of RNA polymerase II transcription occurring from either promoter. Parameters that reduce the amount of transcription from P1 or P2, such as decreased concentration of template injected or decreased incubation time, result in a reduction in the ratio of terminated to read-through c-myc transcripts. Conversely, when transcription levels are increased by higher concentrations of injected template, increased incubation time, or coinjection with competing template, the ratio of terminated to read-through transcripts increases. We hypothesize that an RNA polymerase II processivity function is depleted above a threshold level of transcription initiation, resulting in high levels of premature transcription termination. These findings account for the promoter-specific effects on transcription elongation previously seen in this assay system and suggest a mechanism whereby limiting transcription elongation factors may contribute to transcription regulation in other eukaryotic cells.

Mechanism of replenishment of androgen receptors in cytosol of mouse submandibular gland

1987 ◽  
Vol 115 (3) ◽  
pp. 411-418 ◽  
Author(s):  
S. Kyakumoto ◽  
R. Kurokawa ◽  
M. Ota
Keyword(s):  
Nuclear Receptor ◽  
Actinomycin D ◽  
Androgen Receptors ◽  
Dose Dependence ◽  
Inhibitory Effect ◽  
Nuclear Fraction ◽  
Pretreatment Level ◽  
Receptor Level ◽  
Time Required

ABSTRACT Female mice were used to examine the process of depletion and replenishment of cytosolic androgen receptors in submandibular glands, and to investigate the effects of cycloheximide and actinomycin D on these processes. The dose-dependence of receptor depletion and replenishment in the cytosolic fraction, and that of receptor accumulation in the nuclear fraction were investigated. Almost 100% depletion was revealed 1 h after the injection of testosterone propionate at a dose of 500 or 50 μg testosterone/100 g body weight. With a 5 μg dose, depletion of cytosolic receptors was not complete and replenishment proceeded rapidly compared with that which occurred with the 50 or 500 μg dose. The nuclear receptor level increased 1 h after injection of testosterone, and the raised level was gradually reduced to the pretreatment level with all doses. However, the time required for this return to pretreatment level was dependent on the dose of testosterone. The change in the levels of cytosolic and nuclear androgen receptors following injection of testosterone was parallel to the level of circulating androgen. To determine the requirements for transcriptional and translational events in the replenishment process, cycloheximide and actinomycin D were given in vivo. The process of replenishment of cytosolic receptors was inhibited by the injection of cycloheximide. However, actinomycin D exerted no inhibitory effect on receptor replenishment. Neither cycloheximide nor actinomycin D had any effect on the nuclear receptor level until 6 h after the injection of testosterone. Cycloheximide or actinomycin D alone had no effect on the cytosolic or nuclear receptor level. These results suggest that receptor replenishment involves protein synthesis. J. Endocr. (1987) 115, 411–418

Inhibition of topoisomerase II does not inhibit transcription of RNA polymerase I and II genes

1990 ◽  
Vol 10 (6) ◽  
pp. 2893-2900
Author(s):  
M Dunaway
Keyword(s):  
Rna Polymerase ◽  
Thymidine Kinase ◽  
Topoisomerase Ii ◽  
Xenopus Oocytes ◽  
Initial Period ◽  
Rna Polymerase I ◽  
Time Required ◽  
Polymerase I ◽  
Linear Product

Injection of VM-26 (teniposide) into Xenopus oocytes inhibits the activity of topoisomerase II but does not inhibit transcription by RNA polymerases I and II. A specific assay for topoisomerase II, resolution of catenated DNA molecules into product rings, was used to quantitate VM-26 inhibition in vivo. When catenanes were injected without VM-26, about 60% of them were separated into product rings in the first 5 min after injection, and decatenation of the remainder was complete within 15 min. When VM-26 was coinjected, 60% of the catenanes were separated into product rings in the first 5 min after injection, but the remaining 40% were stable over the next 40 min. At 1 h after injection catenanes were no longer detected in the gel analysis, but the increasing numbers of linear product rings indicated that topoisomerase II continued to be inhibited by VM-26. These results suggest that a short lag of approximately 5 min is required for VM-26 to inhibit topoisomerase II and that after this initial period topoisomerase II is inhibited by more than 90%. There was no detectable decrease in transcription of injected rRNA and thymidine kinase (TK) genes or in the activity of the rRNA enhancer when these transcription templates were coinjected with VM-26. The time required for assembly of injected DNA into chromatin doubled in the presence of VM-26.

scholarly journals A multisite study of performance drivers among institutional review boards

2017 ◽  
Vol 1 (3) ◽  
pp. 192-197 ◽  
Author(s):  
Michael Caligiuri ◽  
Karen Allen ◽  
Nate Buscher ◽  
Lisa Denney ◽  
Cynthia Gates ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Quality Improvement ◽  
Institutional Review Boards ◽  
Federally Funded ◽  
Factors Affecting ◽  
Institutional Review ◽  
Coverage Analysis ◽  
Study Characteristics ◽  
Performance Drivers ◽  
Time Required

IntroductionThe time required to obtain Institutional Review Board (IRB) approval is a frequent subject of efforts to reduce unnecessary delays in initiating clinical trials. This study was conducted by and for IRB directors to better understand factors affecting approval times as a first step in developing a quality improvement framework.Methods807 IRB-approved clinical trials from 5 University of California campuses were analyzed to identify operational and clinical trial characteristics influencing IRB approval times.ResultsHigh workloads, low staff ratios, limited training, and the number and types of ancillary reviews resulted in longer approval times. Biosafety reviews and the need for billing coverage analysis were ancillary reviews that contributed to the longest delays. Federally funded and multisite clinical trials had shorter approval times. Variability in between individual committees at each institution reviewing phase 3 multisite clinical trials also contributed to delays for some protocols. Accreditation was not associated with shorter approval times.ConclusionsReducing unnecessary delays in obtaining IRB approval will require a quality improvement framework that considers operational and study characteristics as well as the larger institutional regulatory environment.

The Critical Point Drying Method Applied to Scanning Electron Microscopy of L-929 Cells

1970 ◽  
Vol 28 ◽  
pp. 278-279
Author(s):  
Charles TurnbiLL ◽  
Delbert E. Philpott
Keyword(s):  
Electron Microscopy ◽  
Carbon Dioxide ◽  
Surface Tension ◽  
Critical Point ◽  
Freeze Drying ◽  
Attractive Alternative ◽  
Crystal Formation ◽  
Critical Point Drying ◽  
Time Required ◽  
Scanning Electron

The advent of the scanning electron microscope (SCEM) has renewed interest in preparing specimens by avoiding the forces of surface tension. The present method of freeze drying by Boyde and Barger (1969) and Small and Marszalek (1969) does prevent surface tension but ice crystal formation and time required for pumping out the specimen to dryness has discouraged us. We believe an attractive alternative to freeze drying is the critical point method originated by Anderson (1951; for electron microscopy. He avoided surface tension effects during drying by first exchanging the specimen water with alcohol, amy L acetate and then with carbon dioxide. He then selected a specific temperature (36.5°C) and pressure (72 Atm.) at which carbon dioxide would pass from the liquid to the gaseous phase without the effect of surface tension This combination of temperature and, pressure is known as the "critical point" of the Liquid.

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