DNA sequence homology between attB-related sites of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and the attP side of γ-Cornephage

Chromosomal restriction fragments of Corynebacterium ulcerans and C. diphtheriae, containing an integration site for corynephages of the beta family, show homology on Southern blots. Homologous DNA in also found in the soil isolate C. glutamicum, although this strain is not susceptible to beta-corynephages. Three of these DNA fragments, one for each bacterial strain, and a fragment of gamma-corynephage DNA previously shown to contain the phage integration site, were cloned and sequenced. Alignment of the 3 bacterial sequences shows a very high degree of homology in a stretch of ca 120 nucleotides, whereas the rest of the sequences is generally non-homologous. Within this common bacterial portion, a segment of ca. 96 nucleotides (core sequence) is also highly homologous to the phage sequence. The first half (ca. 50 bp) of the core sequence is identical in all aligned sequences whereas the second half, which is largely occupied by a stem-and-loop structure, contains point mutations peculiar to each clone. The described sequences are likely to be involved in phage integration/excision processes.     

Interaction of alpha-N-Acetyl-beta-endorphin and calmodulin

Acetylation at the -amino terminal is a common post-translational modification of many peptides and proteins. In the case of the potent opiate peptide -endorphin, -N-acetylation is a known physiological modification that abolishes opiate activity. Since there are no known receptors for -N-acetyl--endorphin, we have studied the association of this peptide with calmodulin, a calcium-dependent protein that binds a variety of peptides, phenothiazines, and enzymes, as a model system for studying acetylated endorphin-protein interactions. Association of the acetylated peptide with calmodulin was demonstrated by cross-linking with bis(sulfosuccinimidyl)suberate; like -endorphin, adducts containing 1 mol and 2 mol of acetylated peptide per mole calmodulin were formed. Some of the bound peptides are evidently in relatively close proximity to each other since, in the presence of amidated (i.e., lysine-blocked) calmodulin, cross-linking yielded peptide dimers. The acetylated peptide exhibited no appreciable helicity in aqueous solution, but in trifluoroethanol (TFE) considerable helicity was formed. Also, a mixture of acetylated peptide and calmodulin was characterized by a circular dichroic spectrum indicative of induced helicity. Empirical prediction rules, applied earlier to -endorphin, suggest that residues 14–24 exhibit -helix potential. This segment has the potential of forming an amphipathic helix; this structural unit is believed to be important in calmodulin binding. The acetylated peptide was capable of inhibiting the calmodulin-mediated stimulation of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity with an effective dose for 50% inhibition of about 3 µM; this inhibitory effect was demonstrated using both an enzyme-enriched preparation as well as highly purified enzyme. Thus, acetylation at the -amino terminal of -endorphin, although abolishing opiate activity, does not interfere with the binding to calmodulin. Indeed, -endorphin and the -N-acetylated peptide behave very similarly with respect to calmodulin association.Portions of this work are in partial fulfillment of the requirements for the Ph.D. degree from Vanderbilt University.     

A new species of Rhynchoidomonas Patton, 1910 (Kinetoplastida: Trypanosomatina) from Operophtera brumata (Lepidoptera: Geometridae)

Summary A new species of Rhynchoidomonas Patton was observed in a single adult male winter moth, Operophtera brumata (L.) from England. Intracellular amastigotes, and extracellular epimastigotes and trypomastigotes with an undulating membrane and free flagellum, were present. All stages had a large, reniform kinetoplast. As transmission of the flagellate between generations of winter moths by ingestion of infected faeces is a virtual impossibility, it is suggested that the flagellate's true host may have been a dipteran parasitoid and that an egg, surface-contaminated with the flagellate, was oviposited into or ingested by a winter moth larva. If the parasitoid had died, this flagellate infection could have been carried over to the adult moth. ac]19830601     

Phenotypic and functional properties of murine thymocytes. III. Kinetic analysis of the recovery of intrathymic cytolytic T lymphocyte precursors after in vivo administration of hydrocortisone acetate

The phenotypic and functional properties of cells in the C57BL/6 mouse thymus regenerating after a single dose of 100 mg/kg hydrocortisone acetate (H/C) are described. Functionally, the frequency of anti-H-2d cytolytic T lymphocyte precursors (CTL-P) in thymuses from individual mice was determined by limit dilution analysis of mixed leukocyte microcultures. The initial increase in CTL-P frequency, seen 48 hr post-H/C, was followed at 6 to 8 days by a phase of rapid decrease. The CTL-P frequency returned to a normal level by 28 days post-H/C. Analysis of the results from individual mice suggested that changes in total thymic CTL-P content were independent of the kinetics of thymus regeneration. Phenotypically, whereas the thymus 48 hr after H/C was considerably depleted of Lyt-2+ cells, there followed a rapid increase in the proportion of such cells to normal levels by 14 days post-H/C. In addition, as measured by FLS, a subpopulation of larger, predominantly Lyt-2+ cells was found during the phase of rapid thymic regeneration. With the use of a monoclonal anti-Thy-1.2 antibody, the weakly Thy-1-staining subpopulation of cells was absent from the thymus at 14 days post-H/C. These changes in the phenotypic properties of the post-H/C regenerating thymus were correlated with changes in their functional properties.     

Studies on the incorporation of labelled sulphate into cells and cell-free extracts of Nitrosomonas europaea

Summary The incorporation of [³⁵S]sulphate was followed into the washed cell suspensions of Nitrosomonas europaea. Thus bound sulphate, sulphite, sulphide, cysteine, glutathione, homocysteine and methionine were found in the ethanol soluble fraction as well as in the residual hydrolysed protein fraction. Cysteic acid, methionine sulphoxide and methionine sulphone were detected in the residual protein. The reaction between sulphydryl groups and N-ethylmaleimide has been successfully used to stabilize the thiol compounds in cell-extracts and the derivatives thus obtained were separated by paper chromatography. As in other microorganisms, sulphate is first activated by ATP in Nitrosomonas before it is reduced. The formation of APS and PAPS has been studied. A pathway for the incorporation of [³⁵S]sulphate is proposed.Abbreviations POPOP 1,4-bis-(5-phenyloxazolyl-2)-benzene - PPO 2,5-diphenyloxazole - APS adenosine-5-phosphosulphate - PAPS adenosine-3-phosphate 5-phosphosulphate - ATP adenosine triphosphate - DNA-ase deoxyribonuclease - NEM N-ethylmaleimide - TCA trichloro-acetic acid - GSH glutathione