Measurement of Methionine-Enkephalin [Arg6,Phe7] in Rat Brain by Specific Radioimmunoassay Directed at Methionine Sulphoxide Enkephalin[Arg6,Phe7]

Abstract: A specific and sensitive radioimmunoassay procedure for Metenkephalin[Arg⁶,Phe⁷] which allows its measurement in regions of the rat brain is described. The antiserum was raised against the methionine sulphoxide derivative of the peptide, and all samples and standards were oxidized with hydrogen peroxide prior to use in the assay with chloramine T-oxidized ¹²⁵I-labelled Met(O)-enkephalin[Arg⁶,Phe⁷]. The only significant cross-reactivity was 30% with the reduced heptapeptide Met-enkephalin[Arg⁶,Phe⁷]. The assay showed less than 0.15% cross-reactivity with fragments of the heptapeptide and with leucine-enkephalin-containing peptides. Acid acetone extraction of rat striatum followed by Sephadex G-50 chromatography and reverse-phase high pressure liquid chromatography showed that essentially all immunoreactivity co-chromatographed with Met-enkephalin[Arg⁶,Phe⁷]. This confirmed the specificity of the assay and showed that the striatum does not contain a high concentration of larger molecular weight forms with the heptapeptide at the COOH terminus. Distribution of the heptapeptide followed that of methionine enkephalin, with highest concentrations in the globus pallidus, intermediate levels in caudate-putamen and hypothalamus, and low levels in cortex and cerebellum.     

Influence of estrogen on glutathione levels and glutathione-metabolizing enzymes in uteri and R3230AC mammary tumors of rats

The glutathione content and the activities of several enzymes in its metabolism, glutathione reductase, glutathione peroxidase and γ-glutamyl transpeptidase, were assayed in uteri obtained from estrogen-treated rats and in R3230AC mammary adenocarcinomas obtained from ovariectomized, intact and estrogen-treated hosts. Normal mammary glands, obtained 10–12 days post-partum, were also examined for these parameters.A daily pharmacological dose of 0.4 μg of estradiol-17β induced a maximal increase in uterine weight and in reduced glutathione (GSH); higher doses of estrogen did not significantly increase either of these parameters. Levels of oxidized glutathione (GSSG) were comparable in both estrogen-treated and untreated rats. The time course of the estrogen-induced uterotrophic response was associated with increases in glutathione reductase, glutathione peroxidase and γ-glutamyl transpeptidase activities with the increased GSH level preceding the increase in uterine weight. Compared to neoplasms from intact or ovariectomized animals, tumors from estrogen-treated hosts exhibited significant decreases in levels of GSSG and GSH, as well as in glutathione reductase and glutathione peroxidase activities, but demonstrated a significant elevation of γ-glutamyl transpeptidase activity. Normal glands from lactating rats had decreased GSH levels, lower activities of glutathione reductase and glutathione peroxidase, but elevated γ-glutamyl transpeptidase activity versus tumors from intact rats. Tumors from estrogen-treated rats more closely resembled mammary glands during lactation. The divergent growth responses elicited by estrogen in the uterus and mammary tumor are correlated with the observed changes in GSH levels and enzymes involved in glutathione metabolism.     

Inhibition of collagen-induced platelet aggregation by antibodies to distinct types of collagens.

Aggregation of platelets by fibrils formed from collagens type I, II and III could be inhibited by coating the fibrils with anti-collagen antibodies or Fab fragments. Similar results were obtained in a clot-retraction assay. Inhibition was achieved with stoichiometric amounts of antibodies and was specific for each type of collagen. Aggregation caused by a mixture of type-I and -III collagens could only be inhibited by a mixture of antibodies against both collagens. The data show that each interstitial collagen is capable of interacting with platelets and do not support the concept of an outstanding activity of type-III collagen.     

The location of highly repetitious DNA in the somatic chromosomes of Drosophila melanogaster

In situ hybridization of Drosophila melanogaster somatic chromosomes has been used to demonstrate the near exact correspondence between the location of highly repetitious DNA and classically defined constitutive heterochromatin. The Y chromosome, in particular, is heavily labeled even by cRNA transcribed from female (XX) DNA templates (i.e., DNA from female Drosophila with 2 Xs and 2 sets of autosomes). This observation confirms earlier reports that the Y chromosome contains repeated DNA sequences that are shared by other chromosomes. In grain counting experiments the Y chromosome shows significantly heavier label than any other chromosome when hybridized with cRNA from XY DNA templates (i.e., DNA from male Drosophila with 1 X and 1 Y plus 2 sets of autosomes). However, the preferential labeling of the Y is abolished if the cRNA is derived from XX DNA. We interpret these results as indicating the presence of a class of Y chromosome specific repeated DNA in D. melanogaster. The relative inefficiency of the X chromosome in binding cRNA from XY and XYY DNA templates, coupled with its ability to bind XX derived cRNA, may also indicate the presence of an X chromosome specific repeated DNA.     

Self-assembly of a plant cell wall in vitro

A method has been developed by which the cell wall of Chlamydomonas reinhardi may be dissociated into its components, and then reassembled in vitro into a product that is chemically and structurally identical to the original cell wall. Chaotropic agents, such as lithium chloride and sodium perchlorate, separate the wall into two fractions, an insoluble amorphous inner wall layer, which retains its integrity (7.5% by weight of the complete wall) and a salt-soluble fraction containing the homogeneous glycoproteins responsible for the outer crystalline layers of the cell wall. Removal of the salt from dissociated walls by dialysis leads to the rapid recovery of complete reassembled cell walls. The conditions necessary for successful reconstitution of the cell wall in vitro include the presence of a suitable surface, across which a decreasing salt gradient exists, and the presence of both the salt-insoluble and the salt-soluble components. The salt-soluble glycoproteins alone can self-assemble under various conditions to form fragments that have the crystalline structure characteristic of the outer layers of the complete cell wall. Both the inner wall layer and the salt-soluble glyco-proteins have similar bulk amino acid and sugar (arabinose, galactose, mannose) compositions and both contain hydroxyproline. On the basis of the in vitro reconstitution of the cell wall we discuss certain aspects of in vivo cell wall morphogenesis. This communication describes the first case in which a plant cell wall has been reconstructed in vitro, and indicates that components of very large cellular structures are capable of being built by a simple self-assembly process.     

Transfer RNA and protein synthesis

The three-dimensional structure of yeast phenylalanine transfert RNA has been determined in orthorhombic crystals. The current status of this work is reviewed together with the relationship of the transfer RNA structure in the crystal to its biologically active form. In addition some speculations are put forward regarding the mode of interaction of tRNA molecules in the ribosome and the manner in which tRNA interacts with aminoacyl synthetase.