Solubilization of receptors for thyrotropin-releasing hormone from GH4C1 rat pituitary cells: demonstration of guanyl nucleotide sensitivity

TRH receptors have been solubilized from GH4C1 cells using the plant glycoside digitonin. Solubilized receptors retain the principal binding characteristics exhibited by the TRH receptor in intact pituitary cells and their membranes. The binding of the methylhistidyl derivative of TRH [( 3H]MeTRH) attained equilibrium within 2-3 h at 4 C, and it was reversible, dissociating with a t1/2 of 7 h. Analysis of [3H]MeTRH binding to soluble receptors at 4 C yielded a dissociation constant (Kd) of 3.8 nM and a total binding capacity (Bmax) of 3.9 pmol/mg protein. Peptides known to interact with non-TRH receptors on GH cells failed to interfere with the binding of [3H]MeTRH, indicating that the TRH binding was specific. Chlordiazepoxide, a competitive antagonist for TRH action in GH cells, inhibited TRH binding to soluble receptors with an IC50 of 11 microM. When [3H]MeTRH was bound to membranes and the membrane proteins were then solubilized, we found enhanced dissociation of the prebound [3H]MeTRH from its solubilized receptor by guanyl nucleotides. Maximal enhancement of [3H]MeTRH dissociation by 10 microM GTP gamma S occurred within about 45 min at 22 C. GTP gamma S, GTP, GDP beta S, and GDP were all effectors of [3H]MeTRH dissociation, exhibiting EC50s in the range of 14-450 nM. The rank order of potency of the tested nucleotides was GTP gamma S greater than GTP congruent to GDP beta S greater than GDP much greater than ATP gamma S greater than GMP. We conclude that TRH receptors have been solubilized from GH cells with digitonin and retain the binding characteristics of TRH receptors in intact pituitary cells. Furthermore, prebinding [3H]MeTRH to GH4C1 cell membranes results in the solubilization of a complex in which the TRH receptor is linked functionally to a GTP binding protein.     

The effects of ultraviolet-B radiation on loblolly pine. I. Growth, photosynthesis and pigment production in greenhouse-grown seedlings

One-year old loblolly pine ( Pinus taeda L.) seedlings were grown in an unshaded greenhouse for 7 months under 4 levels of ultraviolet-B (UV-B) radiation simulating stratospheric ozone reductions of 16, 25 and 40% and included a control with no UV-B radiation. Periodic measurements were made of growth and gas exchange characteristics and needle chlorophyll and UV-B-absorbing-compound concentrations. The effectiveness of UV-B radiation on seedling growth and physiology varied with the UV-B irradiance level. Seedlings receiving the lowest supplemental UV-B irradiance showed reductions in growth and photosynthetic capacity after only 1 month of irradiation. These reductions persisted and resulted in lower biomass production, while no increases in UV-B-absorbing compounds in needles were observed. Seedlings receiving UV-B radiation which simulated a 25% stratospheric ozone reduction showed an increase in UV-B-absorbing-compound concentrations after 6 months, which paralleled a recovery in photosynthesis and growth after an initial decrease in these characteristics. The seedlings grown at the highest UV-B irradiance (40% stratospheric ozone reduction) showed a more rapid increase in the concentration of UV-B-absorbing compounds and no effects of UV-B radiation on growth or photosynthetic capacity until after 4 months at this irradiance. Changes in photosynthetic capacity were probably the result of direct effects on light-dependent processes, since no effects were observed on either needle chlorophyll concentrations or stomatal conductance. Further studies are necessary to determine whether these responses persist and accumulate over subsequent years.     

Immediate breast reconstruction: reducing the risks

One-hundred and sixty-five consecutive immediate breast reconstructions in 157 patients were reviewed. Reconstructions were performed with tissue expanders (53 percent) or immediate gel prostheses (47 percent). Immediate reconstruction was associated with an 18 percent rate of implant loss. Certain risk factors were identified at the p less than 0.05 level using immediate gel implants: failure to achieve complete muscle coverage of the implant, smoking at the time of surgery, initial gel implants of 400 ml or more volume, and age. Expander loss was increased by detaching the pectoralis major (p less than 0.05) and probably by lack of complete muscle coverage in general. Chemotherapy, history of previous smoking, and clinical stage of the carcinoma did not seem to affect reconstructive success. Smoking and patient age should be considered during patient selection for immediate reconstruction. Muscle coverage of the prosthesis should always be attempted. Muscle coverage is mandatory in the smoker. Gel implants of 400 ml or more volume are to be avoided at the initial operation. This approach should enable all surgeons to achieve lower rates of implant loss.     

Expression of a human multidrug resistance cDNA (MDR1) in the bone marrow of transgenic mice: resistance to daunomycin-induced leukopenia.

The human multidrug resistance gene (MDR1) encodes a drug efflux pump glycoprotein (P-glycoprotein) responsible for resistance to multiple cytotoxic drugs. A plasmid carrying a human MDR1 cDNA under the control of a chicken beta-actin promoter was used to generate transgenic mice in which the transgene was mainly expressed in bone marrow and spleen. Immunofluorescence localization studies showed that P-glycoprotein was present on bone marrow cells. Furthermore, leukocyte counts of the transgenic mice treated with daunomycin did not fall, indicating that their bone marrow was resistant to the cytotoxic effect of the drug. Since bone marrow suppression is a major limitation to chemotherapy, these transgenic mice should serve as a model to determine whether higher doses of drugs can cure previously unresponsive cancers.     

Dietary fat influences electric membrane properties of neurons in cell culture

1. SJL/J mice were maintained on semipurified diets which differed in the ratio of polyunsaturated/saturated fatty acid content (P/S). Exposure was from conception and was maintained for periods ranging from 6 to 34 weeks. 2. Neural cell cultures were prepared from dorsal root ganglia (DRG). After 6 and 20 days of culture, neuronal electric membrane properties were determined quantitatively by intracellular recording. 3. A number of significant differences were observed for the two dietary conditions. DRG from mice on the low-P/s diet had an increase in the rate of fall of both phases of repolarization which, in conjunction with the reduced action potential overshoot, led to a reduced action potential duration. This shift to shorter-duration action potentials was accompanied by a shift to more monophasic falling phases. The low-P/S neurons also exhibited a decreased afterhyperpolarization, decreased specific membrane resistance, and decreased membrane electrical time constant compared to high-P/S neurons. 4. It was concluded that the P/S ratio in the diet can have a significant effect on the electric properties of neurons. The high-P/S neurons tended to have action potentials with biphasic repolarizations and longer durations. In contrast, the low-P/S neurons tended to have action potentials with monophasic repolarizations and shorter durations. Moreover, the known ionic dependence of these two types of action potentials suggested that the low-P/S diet resulted in action potentials with a more exclusive Na dependence, while the high-P/S diet resulted in action potentials with both Na and Ca dependence.