Defective excision repair in a mutant of Micrococcus radiodurans hypermutable by some monofunctional alkylating agents

Summary The lethal and mutagenic effects of methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS), and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) can be dissociated in a mitomycin C (MTC)-sensitive mutant, strain 302, of Micrococcus radiodurans.As regards lethality 302 is extremely sensitive, compared with the wild type, to MTC and decarbamoyl MTC (DCMTC), slightly sensitive to EMS, MNNG, nitrous acid, 7-bromomethylbenz {} anthracene (BrMBA), and N-acetoxy-N-2-acetylaminofluorene (AAAF), and resistant to MMS, hydroxylamine, and ICR 191G. As regards mutability it is, compared to the wild type, very sensitive to MMS, EMS, and MNNG, and slightly sensitive to hydroxylamine and nitrous acid but not to any other agent examined.Alkaline sucrose gradient studies indicate that 302 does not incise DNA containing BrMBA adducts, although it does incise DNA damaged by AAAF but probably not to the same extent as wild type.We put forward the hypothesis that the hypermutability of 302 is due to the non-removal of bases or nucleotides, modified in exocyclic positions, which have altered base-pairing capabilities, while lethality results from the non-removal of bases or nucleotides, also modified in exocyclic positions, which no longer form hydrogen-bonded base pairs.     

Vegetative anatomy and morphology of Amentiferae

Some of the important vegetative characters of amentiferous families described in research papers concerned with interfamilial relationships and published since 1947–1948 are briefly summarized. Important conclusions by the writers are also reviewed. Thus, Bataceae are considered derivatives of the Centrospermae ; Betulaceae, Casuarinaceae, and Ulmaceae are hamamelidaceous derivatives; Garryaceae and Cornaceae are closely related; and Julianiaceae and Anacardiaceae are plausible relatives. A more extensive tabulation of vegetative characters of essentially all amentiferous and ranalean families and Dilleniaceae is also presented, and the data are utilized to compare the percentages of advanced and primitive characters in these same taxa. The logic for considering trends whose evolutionary directions were initiated previous or incidental to angiospermous origin as more valid than other evolutionary trends is offered. The following principal conclusions are based upon the analysis of the tabulation: (1) the Amentiferae have sufficient advanced vegetative characters to preclude consideration of them as earliest angiosperms or their direct derivatives unless a large number of primitive features of other sorts offers strong counter evidence; (2) they also have sufficient primitive characters to discount the concept that they are highly evolved; (3) most Amentiferae clearly possess many more advanced vegetative characters than do most Magnoliales, Laurales, Trochodendrales, or Dilleniaceae ; and (4) relationships of several Amentiferae to various, unrelated angiospermous families have been demonstrated.     

Mechanism of Staphylococcus aureus exotoxin A inhibition of Ig production by human B cells

Staphylococcus enterotoxins and toxic shock syndrome toxin 1 are members of a family of exoproteins that are produced by staphylococci and bind specifically to MHC class II molecules. Upon binding to MHC class II molecules, these exoproteins are potent stimulators of T cell proliferation via interaction with specific TCR V-beta segments of both CD4+ and CD8+ T cells. These exoproteins also directly stimulate monocytes to secrete IL-1 and TNF-alpha. Furthermore, these exoproteins have a profound inhibitory effect on Ig production by PBMC. We examined the effects of Staphylococcus enterotoxin A (SEA) on proliferation and Ig production of highly purified human B cells. Our results demonstrated that the binding of SEA to MHC class II molecules on B cells does not alter their ability to proliferate in response to Staphylococcus aureus Cowan strain I (SAC) or to produce Ig in response to SAC plus rIL-2. In contrast, the anti-DR mAb L243 inhibited both B cell proliferation and Ig production. Unable to determine a direct effect of SEA on B cell function, we investigated whether the capacity of SEA to inhibit SAC-induced Ig production by PBMC was T cell-dependent. Our results demonstrated that in the presence of T cells, under appropriate conditions, SEA can either function as a nominal Ag for stimulation of B cell proliferation and Ig production or induce T cell-mediated suppression of Ig production. SEA-induced Ig production required T cell help, which was dependent on pretreatment of the T cells with irradiation or mitomycin C; Ig production was not induced by SEA in the absence of T cells or in the presence of untreated T cells. Furthermore, SEA inhibited Ig production in SAC-stimulated cultures of autologous B cells and untreated T cells; pretreatment of the T cells with irradiation or mitomycin C abrogated SEA-induced inhibition of Ig production. Thus, T cell suppression of SAC-induced Ig production was dependent on T cell proliferation. Similar results were observed with both SEA and toxic shock syndrome toxin 1.     

Solubilization of functional calcitonin receptors.

Calcitonin (CT) binding activity has been extracted from a membrane fraction of human placenta using the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (Chaps). Approximately two-thirds of the available binding sites were extracted using 5 mM-Chaps. The binding characteristics of 125I-labelled salmon CT(125I-sCT) to the solubilized extract were similar to those obtained previously with placental membranes and other targets such as osteoclasts, renal cells and certain human cancer cell lines. 125I-sCT binding was saturable (Bmax. 75 +/- 6 fmol/mg of protein, n = 3) and Scatchard analysis revealed a single class of high-affinity binding sites (Kd 165 +/- 28 pM, n = 3). In competitive-binding studies, various species-specific CTs and CT analogues showed the same rank order of potencies as seen in CT bioassays and several unrelated peptides did not compete at high doses. A biologically active CT analogue, [Arg11,18, Lys14]sCT, derivatized with the photoreactive phenylazide cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate, was used to identify receptor components of Mr approximately 88,000 and approximately 71,000 in both particulate placental membranes and the solubilized extract. Receptor components of Mr 85-90,000 have been identified in other CT target cells previously using chemical- and photoaffinity-labelling techniques. These results demonstrate the first successful solubilization of the CT receptor in a form which purification.     

A proteomics approach to the identification of mammalian mitochondrial small subunit ribosomal proteins

Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins in six individual spots were subjected to in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry. The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled. Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins correspond to Escherichia coli S10 and S14. Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of one of these proteins was observed in D. melanogaster but not in C. elegans, while a homolog of the other was present in C. elegans but not in D. melanogaster. A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome. This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes.     

The resistances of Micrococcus radiodurans to killing and mutation by agents which damage DNA

The resistance of Micrococcus radiodurans to the lethal and mutagenic action 3f ultraviolet (UV) light, ionising (γ) radiation, mitomycin C (MTC), nitrous acid (NA), hydroxylamine (HA), N-methyl-N′-nitro-N-nitrosoguanidine (NG), ethylmethanesulphonate (EMS) and β-propiolactone (βPL) has been compared with that of Escherichia coli B/r.M. radiodurans was much more resistant than E. coli B/r to the lethal effects of UV light (by a factor of 33), γ-radiation (55), NG (15) and NA (62), showed intermediate resistance to MTC (4) and HA(7), but was sensitive to EMS (1) and βPL (2). M. radiodurans was very resistant to mutagens producing damage which can be repaired by a recombination system, indicating that it possesses an extremely efficient recombination repair mechanism.Both species were equally sensitive to mutation to trimethoprim resistance by NG, but M. radiodurans was more resistant the E. coli B/r to the other multagens tested, being non-mutable by UV light, γ-radiation, MTC and HA, and only slightly sensitive to mutation by NA, EMS, and βPL. The resistance of M. radiodurans to mutation by UV-light, γ-radiation and MTC is consistent with an hypothesis that recombination repair in M. radiodurans is accurate since these mutagens may depend on an “error-prone” recombination system for their mutagenic effect in E. coli B/r. However, because M. radiodurans is also resistant to mutagens such as HA and EMS, which are mutagenic in E. coli in the absence of an “error-prone” system, we propose that all the mutagens tested may have a common mode of action in E. coli B/r, but that this mutagenic pathway is missing in M. radiodurans.     

Calcitonin gene-related peptide stimulates cyclic AMP formation in rat aortic smooth muscle cells

In rat aortic smooth muscle cells in culture, calcitonin gene-related peptide stimulated cAMP formation in a dose-dependent manner, half-maximally effective at 0.5 to 1 nM. There was no effect on formation of cGMP, which was increased 300-fold in the same experiments by atriopeptin or sodium nitroprusside. The vasodilator effect of CGRP in rat aorta requires an intact endothelium, indicating that increase in vascular smooth muscle cAMP is not in itself sufficient to bring about relaxation. cAMP is probably a mediator of CGRP action in vascular smooth muscle.     

Na(+) pump alpha 2-isoform specifically couples to contractility in vascular smooth muscle: evidence from gene-targeted neonatal mice

The relative expression of ₁ - and ₂-Na⁺/K⁺-ATPase isoforms found in vascular smooth muscle is developmentally regulated and under hormonal and neurogenic control. The physiological roles of these isoforms in vascular function are not known. It has been postulated that the ₁-isoform serves a "housekeeping" role, whereas the ₂-isoform localizes to a subsarcolemmal compartment and modulates contractility. To test this hypothesis, isoform-specific gene-targeted mice in which the mRNA for either the ₁- or the ₂-Na⁺/K⁺-ATPase isoform was ablated were utilized. Both of these knockouts, and , are lethal; the latter dies at birth, which allows this neonatal aorta to be studied. Isometric force in -aorta was more sensitive to contractile agonists and less sensitive to the vasodilators forskolin and sodium nitroprusside (SNP) than wild-type (WT) aorta; -aortas had intermediate values. In contrast, neonatal -aorta was similar to WT. Western blot analysis indicated a population of 70% ₁- and 30% ₂-isoforms in the WT. Thus in terms of the total Na⁺/K⁺-ATPase protein, the -aorta (at 70%) would be similar to the -aorta (at 65%) but with a dramatically different phenotype. These data suggest that individual -isoforms of the Na⁺/K⁺-ATPase differ functionally and that the ₂-isoform couples more strongly to activation-relaxation pathways. Three-dimensional image-acquisition and deconvolution analyses suggest that the ₂-isoform is distributed differently than the ₁-isoform. Importantly, these isoforms do not localize to the same regions. sodium; potassium; ATPase; contraction; transgenic