“Active” conformation of an inactive semi-synthetic ribonuclease-S

We have studied the integrity of folded structure of a fully active semi-synthetic ribonuclease-S which lacks amino acid residues 16 through 20, and an inactive one with the same residues deleted and 4-fluoro-l-histidine substituted for active site histidine 12. Using “Y” form crystals, we obtained X-ray structural data to a resolution of 2·6 Å and, incorporating phase information calculated from refined ribonuclease-S coordinates, prepared several types of electron density maps. These showed that the overall backbone structure and active site configuration of both analogues do not differ noticeably from those of the native protein. Structural homology extends to the catalytically relevant side-chain at position 12; 4-F-His² assumes the same position as does His in active ribonuclease-S. This supports the view that the 4-F-Hisl2 analogue is inactive due to a change in histidine 12 imidazole basicity, rather than to any significant conformational distortion within the active site.     

Hypophysectomy increases the sensitivity of rats to naloxone-induced hypothermia

Three experiments were done to explore the role of pituitary endorphins in thermoregulation. Hypophysectomized rats were found to be hypothermic when compared to intact rats, but to show a hyperthermic response to handling-induced stress of equal magnitude to that seen in intact animals. In unstressed intact and hypophysectomized animals, the specific opiate antagonist, naloxone, produced a dose-dependent hypothermia, but hypophysectomized animals were found to be ten times more sensitive to naloxone than were the intact animals. In contrast, short-term blockade of pituitary endorphins by dexamethasone had no effect on the magnitude of the naloxone-inducted hypothermia. It was concluded that endorphins of pituitary origin play a long-term tonic role in the modulation of the sensitivity of the brain opioid systems.     

The population ecology of a natural population of the pierid butterfly Colias alexandra

Summary Key factor analysis techniques were used to examine factors determining the abundance of a population of non-pest Colias. The number of individuals entering each successive life stage in the sample population are summarized in life tables for 1975 to 1979. Survivorship to the adult is a relatively consistent proportionality (-x=1.2%, S.D.=1.14; 1975–1979). Factors resulting in reduced natality and, less importantly, mortality during larval diapause determine the population trends for C. alexandra. Egg mortality, pre-diapause larval mortality and postdiapause mortality contribute little to these trends. Possible key sources contributing to reduced natality are examined. Mortality of adults (including removal by collectors), poor weather conditions during the oviposition period, unseasonal snow or drought which affect nectar sources or oviposition sites are among the factors which cause reduced natality and result in population depression.     

Plasmid cloning vectors that can be nicked at a unique site

Summary We describe ColE1-type plasmids, with relaxed DNA replication, based on pMB9, and carrying the Cm^R determinant of R1, in addition to the Tc^R determinant of pMB9. One of the plasmids, pPH207, has unique sites for EcoRI, HindIII, BamI, SalI and Hpal. Insertion of foreign DNA into all but the last of these inactivates cither the Cm^R or the Tc^R determinant.The original Cm^R Tc^R plasmid (pCM2) contains a copy of IS1 which produces deletions to left and to right. Most of these inactivate either the Cm^R or the Tc^R determinant. An internal 280 bp deletion of IS1 DNA in pPH207 greatly reduces the frequency at which deletions are observed.The main feature of these plasmids is a site that is cleaved by some preparations of EcoRI in only one strand of the DNA duplex (the EcoRIⁿ site). This site facilitates strand separation of sequences inserted at the HindIII, BamI and SalI sites of the Tc^R gene, and also of any inserted at the true EcoRI site by a method that destroys that site. Since the oricntation of the EcoRIⁿ site is known, the orientation of sequences inserted at the neighbouring sites can be easily determined.Plasmid pPH207 is not mobilised by a Hfr, but its mobilisation is promoted by ColE1. It is therefore Mob ^- bom ⁺. Experiments with minicells show that it directs the copious synthesis of chloramphenicol transacetylase.     

Liver tissue graft rejection in murine major histocompatibility complex mutants

Liver tissue grafts between seven H-2 mutants and their parental strains have been studied. Each of these mutants was originally identified by reciprocal mutant—parental strain skin graft rejection. However, liver grafts among mutants and parental standard strains are not uniformly rejected. Liver graft rejection also fails to correlate with mutant—parental stimulation in CML and MLC. In addition, the immune reaction pattern of female mutant animals against grafts of male liver differs from the reaction pattern found in parental standard strains. Several explanations for the differences between immune response to liver and skin grafts are proposed, including different T cell subsets involved in recognition, availability of antigenic sites to immunocompetent cells, and structural differences between mutant and parental H-2 antigens. Abbreviations used in this paper: bml, 2, 3, 4,14; dml; fm2=mutants of strains C57BL/6, B10.D2 and B10.M respectively; B6=C57BL/6     

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(1) Cyclic AMP stimulated alanine transport in isolated hepatocytes by approx. 30%, in the range 0.2–5 mM alanine. (2) Alanine utilisation was also stimulated by cyclic AMP. The rates of transport and metabolism were comparable, both in the presence and absence of cyclic AMP. (3) At concentrations of alanine above 1 mM, addition of ouabain, or the reduction of the Na⁺ concentration, could partially inhibit transport without affecting the rate of metabolism. (4) At these alanine concentrations, stimulation of metabolism by cyclic AMP was associated with a decrease in the intracellular to extracellular alanine concentration ratio. (5) At alanine concentrations below 0.5 mM, or at higher concentrations when transport was inhibited by reducing the Na⁺ concentration, cyclic AMP caused an increase in the alanine concentration ratio. (6) It is concluded that at concentrations of alanine above 1 mM, alanine transport is not rate-limiting for alanine metabolism in hepatocytes from fed rats, and cyclic AMP stimulates alanine metabolism primarily by an effect on an intracellular reaction. At physiological concentrations of alanine, however, alanine transport appears to be rate-limiting in agreement with a previous report.     

Separation and properties of prestalk and prespore cells of Dictyostelium discoideum

We describe a method of separating prestalk and prespore cells of Dictyostelium discoideum slugs using a self-generating Percoll gradient. This method gives quantitative recovery of cells and good purity. Separated prestalk and prespore cells possess different levels of the enzymes UDP galactose :polysaccharide transferase, cAMP phosphodiesterase and glycogen phosphorylase. We have used this method, as well as mechanical dissection of slugs, to examine the fate of separated prestalk and prespore cells in Dictyostelium strains that are able to give rise to mature stalk and spore cells in cell monolayers. The results from such experiments provide direct evidence that prestalk and prespore cells from the migrating slug stage are programmed to differentiate into stalk and spore cells respectively.     

Expression of globin genes in teratocarcinoma-Friend cell hybrids

We have used an isoelectric focusing technique to analyse the hemoglobins synthesized by cell hybrids isolated following the fusion of Friend erythroleukemia to embryonal carcinoma cells. Our results confirm that the embryonal carcinoma-derived alpha globin genes are expressed in cell hybrids. In addition, the extent to which the various alpha chains and beta chains are synthesized in these hybrids depends on both the genetic composition of the cell line and on the chemical nature of the agent used to induce hemoglobin synthesis.     

Catabolite repression of different inducible enzymes inEscherichia coli and the effect of cAMP

Simultaneous induction of two enzymes sensitive to catabolite repression does not lead to an additive decrease of the specific activity of the two. Exogenously added cAMP increases the specific activity of catabolically repressed enzymes, irrespective of whether the enzyme is induced separately or simultaneously with another enzyme. In the presence of 12 different substrates metabolized by inducible enzymes glucose does not bring about catabolite repression. Synthesis of cAMP is identical with that occurring under conditions when glucose brings about catabolite repression.