Heat shock mRNA in mouse epidermis after UV irradiation

Total RNA from murine epidermis was extracted at different times after irradiation with erythemogenic doses of ultraviolet light (UVB or UVB/UVA) and hybridized to a DNA probe from the gene of a heat shock protein (hsp 70). An intense and transitory enrichment in RNA molecules hybridizing to the DNA probe was found between 15 and 120 min after irradiation, followed by a return to control levels over the next 70 h. Dose-response analysis indicates that 30 min after the irradiation, the relative amount of RNA hybridizing to the hsp 70 DNA probe increases with the dose (within the range explored: 0-180 mJ/cm2) up to values greater than 5 times the control.     

Histidine modulates the clastogenic effect of oxidative stress

The kinetics of the formation of single-strand breaks induced by H2O2 in DNA is more rapid in Eagle's Modified Minimal Essential Medium (MEM) than in phosphate-buffered saline (PBS). Among the components of MEM, we found that histidine increases the rate of degradation of DNA by H2O2 in PBS dose-dependently. In hamster lung fibroblasts histidine increases the cytotoxicity of H2O2, as well as the number of sister-chromatid exchanges and the frequency of micronuclei induced by hydrogen peroxide.     

Liposome-delivered phosphatidylcholine enhances the acetylcholine sensitivity of dystrophic mouse myotubes

Myotubes were obtained in vitro from satellite cells of normal and dystrophic C57BL/6J/dydy mice. The acetylcholine sensitivity (mV/nC) of dystrophic myotubes determined with conventional electrophysiological techniques, was lower than that of normal myotubes. Incubation of dystrophic myotubes with liposomes containing phosphatidylcholine (a lipid present in higher amounts in normal adult muscle) significantly increased their acetylcholine sensitivity.     

Antipeptide antibodies to the 141-1141-1141-1receptor confirm the extracellular orientation of the amino-terminus and the putative first extracellular loop

Summary We developed site-directed rabbit antisera against synthetic peptides selected from the deduced amino acid sequence of the hamster lung ₂-adrenergic receptor (amino acids 16–31 and 174–189, respectively). All antisera directed against peptide 1 (four of four rabbits) as well as two antisera directed against peptide 2 (two of four rabbits) recognized the purified ₂-adrenergic receptor in immunoblot conditions when used at a dilution of 1500. Antisera directed against peptide 1 as well as peptide 2 were able to immunoprecipitate iodinated as well as¹²⁵I-cyanopindolol tabeled ₂-adrenergic receptor. This last result implies that the recognized epitopes do not contain the¹²⁵I-cyanopindolol binding domain of the ₂-adrenergic receptor. Immunoblot experiments performed on membrane fractions from hamster lung tissue showed that immunoreactive bands at 64,000, 57,000, 47,000, 44,000 and 38,000 daltons were specifically detected. When purified ₂-adrenergic receptor was iodinated and submitted to glycolytic and/or tryptic treatments, species with similar molecular weights could be recovered. Then, the immunoreactive bands probably correspond to native ₂-adrenergic receptor and to degradative or nonglycosylated species of this molecule. The antisera were also able to detect immunoreactive molecules in murine and human cell lines, suggesting conservation of the probed sequences between these species. Enzymatic linked immunosorbent assay tests on intact cells and immunofluorescence studies confirmed that the amino-terminus and putative first extracellular loop are extracellularly located. Immunofluorescence studies on mouse brain primary cultures showed that cells expressing ₂-adrenergic receptor-like molecules exhibited a neuronal phenotype.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Stimulates Adenylyl Cyclase and Phospholipase C Activity in Rat Cerebellar Neuroblasts

Abstract: The presence of receptors for the novel neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been recently demonstrated in the external granule cell layer of the cerebellum, a germinative matrix that generates the majority of cerebellar interneurons. In the present study, we have taken advantage of the possibility of obtaining a culture preparation that is greatly enriched in immature cerebellar granule cells to investigate the effect of PACAP on the adenylyl cyclase and phospholipase C transduction pathways. The two molecular forms of PACAP, i.e., 27-(PACAP27) and 38-(PACAP38) amino-acid forms of PACAP, induced a dose-dependent stimulation of cyclic AMP production in granule cells. The potencies of PACAP27 and PACAP38 were similar (ED₅₀ = 0.12 ± 0.01 and 0.23 ± 0.07 n M , respectively), whereas vasoactive intestinal polypeptide (VIP) was ∼100 times less potent. PACAP27 and PACAP38 also induced a dose-dependent stimulation of polyphosphoinositide breakdown (ED₅₀ = 19.1 ± 6.3 and 13.4 ± 6.0 n M , respectively), whereas VIP had no effect on polyphosphoinositide metabolism. The effect of PACAP38 on inositol phosphate formation was significantly reduced by U-73122 and by pertussis toxin, indicating that activation of PACAP receptors causes stimulation of a phospholipase C through a pertussis toxin-sensitive G protein. In contrast, forskolin and dibutyryl cyclic AMP did not affect PACAP-induced stimulation of inositol phosphates. Taken together, the present results demonstrate that PACAP stimulates independently the adenylyl cyclase and the phospholipase C transduction pathways in immature cerebellar granule cells. These data favor the concept that PACAP may play important roles in the control of proliferation and/or differentiation of cerebellar neuroblasts.     

The role of proline residues in the folding kinetics of the bovine pancreatic trypsin inhibitor derivative RCAM(14–38)

The role of proline residues in the folding of the trypsin inhibitor derivative RCAM(14–38) has been studied by testing for slow-folding species of the unfolded protein, which could result from the introduction of wrong proline isomers after unfolding. The unfolded protein at 25 °C contains chiefly fast-folding (U_F) molecules: they refold with a time constant of 40 milliseconds at pH 6.8 in 1.9 m-guanidinium chloride. At least one minor slow-folding (U_s) species has been found, using fluorescence to monitor refolding. The reaction in which this U_s species is formed after unfolding shows the properties expected for the cis: Irans isomerization of a proline residue. When refolding is monitored by tyrosine absorbance, two minor slow reactions are found. The faster reaction is in the same time range (15 s at 25 °C) as that studied by fluorescence, and the slower reaction is quite slow (200 s at 25 °C). It is not known whether the slower reaction results from a second U_s species. There are four trans proline residues in bovine pancreatic trypsin inhibitor: the proportion of slow-folding molecules (not more than 25% at 25 °C) is smaller than expected if every proline residue can produce a U_s species and if the cis to trans ratio of each residue after unfolding is at least 0.1:0.9.Criteria based on folding kinetics are given for classifying the types of folding reaction shown by unfolded molecules containing a single wrong proline isomer. Levitt (1980) has classified three types of proline residues according to the energy difference (small, intermediate or large) between the native protein and the predicted minimum energy structure containing a wrong proline isomer. He suggests that these three types of proline residues can be recognized by the types of folding reactions they produce. Only type II (intermediate) folding reactions have thus far been characterized by the criteria introduced here. We point out that the type of folding reaction depends also on the folding conditions, and a possible explanation for this effect is given.     

Protein . nucleic-acid reaction kinetics. Theoretical analysis of the binding reaction between DNA and RNA polymerase.

This paper presents methods developed in order to analyze experimental results concerning the binding of Escherichia coli DNA-dependent RNA polymerase to DNA at high and at low DNA concentrations, using the filter retention assay. The basis hypotheses, under which the mathematical expressions for describing the kinetics of binding are derived, are as follows. (a) At low DNA concentration: equivalence and independence of the specific binding sites; first-order dependence of the binding reaction on both DNA and protein concentration. (b) At high DNA concentration: equivalence and independence of the non-specific binding sites; no direct transfer or one-dimensional sliding of the protein along the DNA. Comparison between theoretical predictions and experimental results at high DNA concentration will allow one to determine the relative value of the rates of binding of RNA polymerase to different promoters (between 1 and 2 in T5 DNA). Binding experiments performed at low DNA concentration are reported in this paper: these results and the analysis which is reported allow one to determine the value of the rate constant of formation of non-filterable complexes for the system fd DNA (replicative form) . RNA-polymerase (kappa a = 3.3 X 10(8) M-1 s-1 in 0.1 M NaCl, 0.01 M MgCl2).     

Disorders of the Menstrual Cycle in Elite Female Ice Hockey Players and Figure Skaters

The purpose of this study was to assess and compare the incidence of delayed menarche and menstrual dysfunction among elite ice hockey players and figure skaters. Forty-three ice hockey players (23.5 ± 4.8 years, 68.2 ± 1.2 kg, 1.68 ± 0.01 m) and 39 figure skaters (17.5 ± 3.4 years, 53.7 ± 5.8 kg, 1.64 ± 0.05 m) completed a self-administered questionnaire on their menstrual status and history, training regimens and lifestyle. Age at menarche did not differ significantly between ice hockey players (13.3 ± 1.3 years) and figure skaters (13.7 ± 1.4 years). Menarche was unrelated to nationality, vigorous training premenarche or age at which the athlete began her sport, but was correlated with the age at menarche of the athletes’ mothers (r = 0.39, p < 0.05). Hormonal contraceptives were used by 35% of ice hockey players and 15% of the figure skaters. Amenorrhea and oligomenorrhea were experienced by 7.1% and 38.7% of postmenarcheal, ice hockey players and figure skaters respectively not using hormonal contraceptives. Menstrual dysfunction was associated with both age and age at menarche in the ice hockey players only. Training factors, and psychological pressure were perceived by the athletes to contribute to menstrual dysfunction. A greater training volume, younger age at commencing sport, lower body mass, greater subjective body image pressure and younger biological and gynaecological age were reported among the figure skaters, and were proposed to explain the higher incidence of menstrual dysfunction among the figure skaters compared with the ice hockey players. Figure skaters appear at increased risk of amenorrhea and oligomenorrhea compared with ice hockey players, which may be linked to training and physical characteristics of the sports.