Kinetic properties of cholinergic desensitization in Aplysia neurons

The kinetic properties of desensitization onset of excitatory cholinergic responses were studied in isolated, voltage-clamped Aplysia neurons. Desensitization of the acetylcholine (ACh)-induced current in response to microperfused acetylcholine occurred in two phases, and was best modelled as the sum of two exponential components plus a constant. Both exponential components were accelerated by increasing ACh dose. At the higher ACh doses the current decline was dominated by the fast exponential component, and the ratio of the plateau-peak current was reduced. Over the range of membrane potentials -50 to -110 mV, no change in the kinetics of desensitization onset was observed. The mean time constants of both exponential components were doubled by cooling from 20 degrees C to 5 degrees C. These results demonstrate that, as at the vertebrate neuromuscular junction, the onset of desensitization of this ACh response involves at least two processes which are dose- and temperature-sensitive. The lack of voltage dependence contrasts with results from vertebrate preparations, and indicates a fundamental difference between the properties of the excitatory ACh response in Aplysia neurons and the vertebrate neuromuscular junction.     

Isolation of an evolutionarily conserved epidermal growth factor receptor cDNA from human A431 carcinoma cells

Complementary DNA corresponding to total poly(A)+-RNA from the human A431 epidermoid carcinoma cell line was cloned in the phage expression vector lambda gt 11. An epidermal growth factor (EGF) receptor cDNA clone was obtained by screening of the expression library with a rabbit polyclonal antibody (IgG), raised to the purified A431 EGF receptor, in combination with [125I]protein A of S. aureus. The cloned cDNA was able to select, by hybridization, messenger RNA which was translated in Xenopus oocytes and yielded an immunoprecipitable EGF receptor protein of Mr = 160,000. The insert of this cDNA (phEGFR-1), is approximately 880 base pairs in length and encodes the carboxyterminal portion of the EGF receptor protein. Its sequence is evolutionarily conserved among vertebrates as shown by hybridization to unique chromosomal DNA sequences from human, baboon, dog, rat, mouse and frog.     

Flow cytometric determination of radiation-induced chromosome damage and its correlation with cell survival

Chinese hamster M3-1 cells were irradiated with several doses of X rays or alpha particles from 238Pu. Propidium iodide-stained chromosome suspensions were prepared at different times after irradiation; cells were also assayed for survival. The DNA histograms of these chromosomes showed increased background counts with increased doses of radiation. This increase in background was cell-cycle dependent and was correlated with cell survival. The correlation between radiation-induced chromosome damage and cell survival was the same for X rays and alpha particles. Data are presented which indicate that flow cytometric analysis of chromosomes of irradiated cell populations can be a useful adjunct to classical cytogenic analysis of irradiation-induced chromosomal damage by virtue of its ability to express and measure chromosomal damage not seen by classical cytogenic methods.     

Subchromosomal DNA synthesis in synchronous V-79 chinese hamster cells after X irradiation

A substantial fraction of replicon initiation events in Chinese hamster V-79 cells have been shown to be refractory to the effects of X irradiation immediately after exposure. This study examines the possibility that the initiation radiorefractive portion is the result of changes in replicon radiosensitivity as a function of position in S phase. The data obtained from DNA fiber autoradiograms and kinetic incorporation of radiolabeled thymidine from cells irradiated at various positions in S phase showed only slight changes in the proportion of replicons refractive to X irradiation immediately after exposure. These results indicate that initiation radiorefractive replicons may be an intrinsic property of V-79 cells and that cell-cycle-specific heterogeneity in radiation response cannot fully account for this phenomenon. The results also indicate that delayed inhibition of initiation events may play a larger role in the observed radiorefractive fraction than previously thought.     

N-methyl-d-aspartate andl-aspartate activate distinct receptors in piriform cortex

The effects of ionophoretically applied N-methyl-DL-aspartate (NMDA) and aspartate on identified pyramidal neurons in rat piriform cortex were examined in isolated, submerged, and perfused brain slices. NMDA was more potent than aspartate in eliciting neuronal discharge. Perfusion of the acidic amino acid antagonists, DL-2-amino-5-phosphonovalerate (APV), 10(-6) or 10(-5) M, DL-2-amino-7-phosphonoheptanoate (APH), 10(-5) M, and gamma-D-glutamylglycine (gamma DGG), 10(-5) M, selectively blocked the response to NMDA without effect on the response to aspartate. At higher concentrations which blocked responses to both NMDA and aspartate, gamma DGG blocked kainate responses and depressed glutamate and quisqualate responses. These results suggest that in piriform neurons NMDA and aspartate act at distinct receptor sites, not a common receptor site, and that both of these sites are distinct from those that mediate responses to glutamate, quisqualate, and kainate.     

High-level expression in Escherichia coli of calcium-binding domains of an embryonic sea urchin protein

A plasmid expression vector is described having features that facilitate high-level expression of eukaryotic DNA in Escherichia coli. The vector, designated pMAM17, carries the ColE1 rop gene under the control of the thermally inducible lambda PL promoter. The rop gene product is a negative regulator of ColE1 DNA replication, and its high-level expression is lethal to cells. However, cells harboring a plasmid with an insert in the rop gene grow normally under these conditions. pMAM17 has been used to investigate the properties of a family of proteins expressed in the dorsal ectoderm of sea urchin embryos. The coding sequences of these proteins (termed Spec proteins) have homology to the troponin C superfamily. Large amounts of the Rop-Spec fusion protein were produced at 42 degrees C in E. coli. Unfractionated E. coli extracts containing the fusion protein could be used to produce antibodies that were highly specific for Spec proteins present in crude extracts of sea urchin embryos. Analysis of the Rop-Spec fusion protein on SDS-polyacrylamide gels in the presence and absence of EGTA indicated that the fusion protein bound calcium ions in a manner characteristic of proteins of the troponin C superfamily. This behavior provides biochemical evidence that the Spec proteins are functionally homologous to other members of this superfamily.     

Mapping of the structural gene for the second component of complement with respect to the human major histocompatibility complex.

Families have been HLA typed, and allotypes of the second component of complement and properdin factor B determined. The lod score for the C2 structural gene and HLA-B from the study of 11 families and 55 informative meioses was 14.39 at maximum likelihood estimate of the recombination fraction of .02. This is related to other estimates of the distance between these two genes. The relative kinetic activities of the C2 allotypes were studied and no differences were demonstrated. No crossovers between Bf and C2 were observed.     

Effect of the fluctuations in electron density within a globular protein on the excess small-angle X-ray scattering

Excess small angle X-ray scattering in solvents of differing electron density has been calculated from the crystal structures obtained for rubredoxin, trypsin inhibitor, myogen, ferricytochrome c₂, ribonuclease S, lysozyme, nuclease, myoglobin, α-chymotrypsin, elastase, subtilisin, carboxypeptidase A, thermolysin, methemoglobin, deoxyhemoglobin, and a single polypeptide chain of M₄ lactate dehydrogenase. The scattering curves for each protein can be reproduced by the sum of three curves, with the weighting of the three curves depending on the electron density of the solvent. The radius of gyration obtained from the small angle X-ray scattering by globular proteins in aqueous solution will usually exceed the values defined by the shape of the macromolecule. Deviations for certain of the proteins cited are calculated to be as large as 6%. These deviations arise from the tendency for the amino acid residues with low electron density to be situated closer to the center of the protein than the amino acid residues of high electron density. An upper limit of 19% is obtained for the discrepancy between the radius of gyration defined by the shape of a spherical globular protein of typical amino acid composition and the apparent radius of gyration measured for that protein in water by small angle X-ray scattering.