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D Michael

Publications and source records attributed to D Michael.

14 recordsLinked to original sources
PubMed1992

Zinc (Zn2+) blocks voltage gated calcium channels in cultured rat dorsal root ganglion cells.

Dorsal root ganglion cells (DRGs) exhibit 3 types of voltage-dependent calcium channels. We have cultured DRGs from 2- to 4-day-old rat pups and obtained whole-cell patch-clamp recordings of calcium-channel currents after 1-5 days in culture. The calcium-channel currents (carried by barium) were recorded with tetrodotoxin (TTX) in the external solution. A cesium-based solution containing Na-ATP, HEPES and EGTA was used in the recording pipette. Cells were held at -80 mV and calcium channel currents were evoked by stepping to depolarized voltages. The divalent cation zinc (Zn2+) blocked sustained and transient voltage sensitive calcium channel currents. Onset of the blockade was fast and a steady-state was reached within 5-15 min, depending upon the concentration used. The IC50 for inhibition of the peak current evoked by a step depolarization from -80 mV to 0 mV (N plus L channels) for 80 ms was 69 microM Zn2+ and the Hill slope about 1. The calcium current evoked by a voltage step from -80 mV to voltages between -40 mV and -15 mV (T-type current) was more sensitive (> 80% block with 20 microM Zn2+). During wash the effect was only partly reversible in 50% of the neurons. Thus, Zn2+ is a potent blocker of voltage dependent calcium currents in mammalian neurons, especially of T-type currents.

Animals↗
PubMed1991

Effect of differential saturation on the spatial localization performance of depth pulses.

Computer simulations of Depth pulse B (theta; (2 theta [+/- x, +/- y])2; acquire) and other Depth pulses, verified by experimental surface coil NMR studies utilizing phantom samples, reveal that the spatial localization performance of Depth pulses degrades when the repetition time is short relative to T1 because of differential saturation, i.e. T1 discrimination effects. Simulations of Depth pulse A (theta; 2 theta [+/- x, +/- y]; acquire) and Depth pulse B indicate that there is no phase-cycled pulse sequence delivery order which negates the untoward effect of T1 discrimination on spatial localization performance. The results of this study demonstrate the importance of consistent magnetization preparation prior to the delivery of each phase-cycled multiple pulse sequence in a Depth pulse cycle for obtaining optimal spatial localization performance. The untoward effects of inconsistent magnetization preparation, resulting from T1 discrimination, may be ameliorated by the application of many Depth pulse cycles.

Computer Simulation↗
PubMed1990

Assessment of protein reorientational diffusion in solution by 13C off-resonance rotating frame spin-lattice relaxation: effect of anisotropic tumbling.

The 13C off-resonance rotating frame spin-lattice relaxation technique is applicable to the study of protein rotational diffusion behavior in a variety of experimental situations. The original formalism of James and co-workers (1978) (J. Amer. Chem. Soc. 100, 3590-3594) was constrained by the assumption of random isotropic reorientational motion. Here we include in the formalism anisotropic tumbling, and present the results of computer simulations illustrating the differences between anisotropic and isotropic reorientational motion for the off-resonance rotating frame spin-lattice relaxation experiment. In addition, we have included chemical shift anisotropy of the peptide carbonyl carbon as an additional relaxation mechanism contribution, to permit high-field nmr protein rotational diffusion measurements.

Carbon Isotopes↗
PubMed1987

Response of carcinogen-altered mouse epidermal cells to phorbol ester tumor promoters and calcium.

Primary cultures of mouse epidermal cells are induced to terminally differentiate when extracellular calcium levels are increased to more than 0.1 mM. After carcinogen treatment, cellular foci can be selected that resist this calcium signal to terminally differentiate. Calcium causes these foci to stratify; however, in contrast to normal epidermis, DNA-synthesizing cells in these foci are found in the suprabasal cell layers as well as in basal cells. Cell lines derived from these foci may be considered to be putative initiated cells. Three of these cell lines, designated 308, D, and F, have been characterized for their response to calcium and phorbol ester tumor promoters. The formation of cornified cells and the activity of epidermal transglutaminase were utilized as markers of epidermal differentiation. Neither calcium nor the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increased transglutaminase activity or cornification of any of the 3 lines. Proliferation was estimated by the [3H]thymidine labeling index, by incorporation of [3H]thymidine into DNA, and by a clonal growth assay. Unlike primary normal cultures, raising the calcium level of the medium did not markedly reduce the rate of proliferation of any of the 3 cell lines. In 2 of the lines, line 308 and line D, proliferation increased in response to TPA exposure. In line F, [3H]thymidine incorporation in confluent cultures was inhibited by TPA, while in cells plated at clonal densities, TPA was cytotoxic at doses of 5 ng/ml or higher. If these calcium-resistant epidermal cell lines correspond to initiated cells, their lack of sensitivity to the induction of terminal differentiation by TPA could account for their growth relative to normal cells. Those lines that also respond to stimulation of proliferation by TPA to a greater extent than normal cells would have a further growth advantage.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1986

Digital zero-phase-shift filtering of short-latency somatosensory evoked potentials.

Short-latency somatosensory potentials evoked by median nerve stimulation were averaged after analogue bandpass of 30-3000 Hz and then filtered by a zero-phase-shift digital filter with bandpass of 200-1500 Hz. Both near- and far-field potentials recorded by scalp to scalp, ear and extracephalic references were enhanced, without temporal distortion or the introduction of spurious peaks. Components were identified at the same or similar peak latencies as reported by other investigators. Zero-phase-shift digital filtering may increase the usefulness of recording short-latency somatosensory potentials.

Adult↗
PubMed1986

Cultivation and characterization of cells derived from mouse skin papillomas induced by an initiation-promotion protocol.

Methods to culture cells from papillomas induced by an initiation-promotion protocol in SENCAR mice were developed, and the resultant cell lines have been characterized. Using Eagle's medium with 0.05 mM Ca2+ conditioned by dermal fibroblasts and supplemented with 1 ng/ml epidermal growth factor (EGF) in culture dishes coated with collagen and fibronectin, six cell lines (PA, PB, PC, PD, PE and PF) were established from separate pools of papillomas. When tested for tumorigenicity in nude mice by injection of a cell suspension or implantation of cells growing on a plastic liner, two of the lines (PC and PF) produced no tumors at any passage. In contrast, cells of the lines PA and PE produced highly differentiated squamous cell carcinomas from the earliest passage tested. The results with PB and PD were variable on tumorigenicity testing with some passages positive and others negative. When tested for responsiveness to Ca2+ (greater than 0.1 mM) as a differentiation stimulus, all lines responded. In the higher Ca2+ medium there was a 50-95% decrease in colony-forming efficiency, a slight decrease in [3H]thymidine incorporation (except for PA) and an increase in the number of cornified cells (except for early passage PF). Epidermal transglutaminase activity, a marker for terminal differentiation, was increased in the presence of medium with Ca2+ greater than 0.1 mM. However, unlike normal cells, only a fraction of the cells from each of the papilloma-derived cell lines terminally differentiated in response to Ca2+ while the remaining cells continued to proliferate, although at a slower rate. Responsiveness to phorbol ester tumor promoters was also examined in papilloma cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment increased colony forming efficiency, DNA synthesis and colony size in all lines in medium with either 0.05 mM Ca2+ or 1.2 mM Ca2+. TPA treatment also increased ornithine decarboxylase activity in all lines, even at the higher Ca2+ concentration, although normal keratinocytes respond only when grown in medium with low Ca2+. TPA treatment caused only a slight increase in the number of cornified cells and no increase in epidermal transglutaminase activity in papilloma cells while it causes 10-fold or greater increases in these differentiation markers in normal keratinocytes. Thus papilloma cells appear to differ from normal keratinocytes in their ability to maintain a proliferating population under conditions favoring terminal differentiation, their consistent proliferative response to phorbol esters under these same conditions, and their reduced sensitivity to phorbol ester-induced terminal differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1976

Natural synchrony of newborn mouse epidermal cells in vitro.

Epidermal cells were separated from newborn mouse skin and grown on glass chamber slides or in plastic Petri dishes. Cell proliferation was examined at short intervals during the first 3 to 4 days in culture. DNA synthesis was estimated by the incorporation of [3H]thymidine into epidermal DNA, or by the labeling index. The mitotic rate was estimated by blocking mitotic cells with vinblastine. Reasonable agreement was found with the different methods. The cells grew synchronously with peaks of DNA synthesis at 21 to 23 hr, 35 to 40 hr, and 60 hr; peak mitotic rates were seen at 44 to 48 hr and 72 to 76 hr. The cells appeared to grow exponentially during the first 3 to 4 days in culture, with one main cohort of cells dividing during the successive proliferative peaks.

Animals↗
PubMed1976

Guanine-specific DNA repair after treatment of mouse skin cells with N-methyl-N'-nitro-N-nitrosoguanidine.

The incorporation of [3H]thymidine and [3H]deoxyguanosine into DNA was studied during DNA repair in mouse skin cells treated with the skin tumor initiator N-methyl-N'-nitro-N-nitrosoganidine. At high, toxic levels of N-methyl-N'-nitro-N-nitrosoguanidine, repair (incorporation of the precursor into DNA which had not replicated) was demonstrated with both precursors. At lower, less toxic doses of the carcinogen, repair could not be demonstrated with [3H]-thymidine, but it was clearly demonstrable with [3H]deoxyguanosine. Thus, we are apparently observing two kinds of DNA repair, one in which a single base (in this case, guanine) replaces a base lost by chemical or enzymatic depurination and the second in which more than one base is replaced, indicating synthesis of longer stretches DNA after extensive enzymatic excision. The guanine-specific repair shown at relatively nontoxic dose levels of N-methyl-N'-nitro-N-nitrosouanidine may be more relevant to the survival of cells than the repair demonstrated with [3H]thymidine at higher doses.

Animals↗
PubMed1976

Dimethyl sulfoxide-induced enhancement of 7,12-dimethylbenz(a)anthracene metabolism and DNA binding in differentiating mouse epidermal cell cultures.

Mouse epidermal cells in primary culture differentiate rapidly over a 2-week period leading to keratinization and sloughing of most of the plated cells. Cell replication was partially synchronized in these cultures with peaks of DNA synthesis at the 2nd and 8th day. The ability of epidermal cells to metabolize 7,12-dimethylbenz[a]anthracene and the subsequent binding of activated products to epidermal DNA was a function of the culture time. Constitutive and induced levels of aryl hydrocarbon hydroxylase in cells cultured for 10 days were half of those in cells grown for 3 days. Likewise, 7,12-dimethylbenz[a]anthracene binding to epidermal DNA was two- to fourfold lower in 10-day than 3-day cultures. This decrease in metabolism and binding between 3-day and 10-day cultures could be eliminated by the inclusion of 1.25% dimethyl sulfoxide in the culture medium during the entire culture period.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1976

Stimulated DNA synthesis in mouse epidermal cell cultures treated with 12-O-tetradecanoyl-phorbol-13-acetate.

Exposure of mouse epidermal cells in culture to 12-O-tetradecanoyl-phorobol-13-acetate (TPA) results in an initial inhibition of DNA synthesis for 24 hr followed by a 5- to 10-fold stimulation at 72 to 96 hr. A corresponding increase in mitotic rate also occurs at 72 to 96 hr. These responses occur when TPA is continuously present in the medium or if the exposure is as short as 1 hr, but the degree of stimulation was dependent on dose and duration of exposure. Sensitivity to TPA varied with the length of time the cells were in culture prior to treatment. TPA treatment also produced an alteration in morphology from clearly epithelial to a more fibroblastic type. These biochemical and morphological effects did not occur after treatment of epidermal cells with either phorbol-13,20-diacetate or phorbol. Primary dermal fibroblasts in culture did not respond to TPA in this manner, but a line of cultured liver epithelial cells was slightly stimulated by the promoter. This system appears to be a sensitive in vitro model for detecting the hyperplasia-inducing effects of phorbol esters and should be used for mechanistic studies and bioassay.

Animals↗