Poly(adenosine diphosphoribose) synthesis in ultraviolet-irradiated xeroderma pigmentosum cells reconstituted with Micrococcus luteus UV endonuclease

Synthesis of DNA and poly(adenosine diphosphoribose) [poly(ADPR)] was examined in permeabilized xeroderma pigmentosum lymphoblasts (XP3BE) before and after UV irradiation and in the presence and absence of Micrococcus luteus UV endonuclease. M. luteus UV endonuclease had no effect on the level of DNA or poly(ADPR) synthesis in control, unirradiated cells. UV irradiation caused a decrease in replicative DNA synthesis without any significant change in poly(ADPR) synthesis. In UV-irradiated cells treated with M. luteus UV endonuclease, DNA synthesis was restored to a level slightly greater than in the unirradiated control cells, and poly(ADPR) synthesis increased by 2- to 4-fold. Time--course studies showed that the UV endonuclease dependent poly(ADPR) synthesis preceded the endonuclease-dependent DNA synthesis. Inhibition of endonuclease-dependent poly(ADPR) synthesis with 3-aminobenzamide, 5-methylnicotinamide, or theophylline produced a partial inhibition of the endonuclease-dependent DNA synthesis. Conversely, inhibition of the endonuclease-dependent DNA synthesis with dideoxythymidine triphosphate, phosphonoacetic acid, or aphidicolin had no effect on the endonuclease-dependent poly(ADPR) synthesis. These studies show that stimulation of poly(ADPR) synthesis in UV-irradiated cells occurs subsequent to the DNA strand breaks created by the specific action of the UV endonuclease on UV-irradiated DNA. The effect of the inhibitors of poly(ADPR) synthesis in UV-irradiated cells indicates that the endonuclease-stimulated DNA synthesis is dependent in part on the prior synthesis of poly(ADPR).     

Native C3 does not bind to the C3b receptor (CR1) of human blood B lymphocytes or alter immunoglobulin synthesis

Complement receptors on lymphocytes were first described more than 12 yr ago (1-3) and have come to be used as a common marker for the identification of B cells (4). The function of these receptors on the lymphocyte and their possible role in induction and/or regulation of the immune response remain unclear. In particular, there continues to be controversy as to whether native C3 can bind to the C3b receptor of these cells without cleavage to C3b (5-10). The resolution of this question is critical in order to clarify the expected state of availability of the receptor in vivo, because in plasma, the C3 concentration is relatively high (1.1 to 1.5 mg/ml), whereas there is little or no circulating C3b due to efficient degradation by factor H and the C3-inactivator (11). With the recent development of an improved method for the isolation of C3 from human plasma, it has been possible to obtain biochemically and functionally pure C3 that has not undergone structural or conformational alteration during processing and fully retains the specific hemolytic activity of C3 in fresh serum (12). Berger et al. (13) were able to demonstrate that C3 prepared in this way failed to bind to the C3b receptor of human polymorphonuclear leukocytes or erythrocytes. Similar observations were made by Schreiber et al. (14), also with phagocytic cells and erythrocytes, and by Dixit et al. (15) with an isolated membrane receptor preparation from rabbit macrophages. In the present communication, we extend these observations to human peripheral blood B lymphocytes. Purified C3 in its native state fails to block B lymphocyte-EA (IgM) C4b3b rosettes, whereas C3b causes 50% inhibition at 5 to 6 micrograms/ml. Furthermore, C3 failed to alter polyclonal immunoglobulin (Ig) production by human B cells, whereas C3b inhibited this B cell function. These data suggest that native C3 does not bind to the C3b receptors of B lymphocytes, and thus they are not occupied under normal conditions in vivo.     

Olfactory function in patients with hypogonadotropic hypogonadism: an all-or-none phenomenon?

Hypogonadotropic hypogonadism (HH) refers to an endocrine defectof hypothalamic origin resulting in gonadal hypoplasia and frequentlyassociated with anosmia or severely impaired olfactory function(Kallmann's syndrome). This apparently results from a disruptionin the migration of neurons from the olfactory placode to thebulb and hypothalamus early in development, and so providesa unique opportunity to investigate olfactory function in humansubjects with congenitally incomplete peripheral systems. Olfactoryperformance in 37 HH patients and 37 age-matched controls wascompared using a modified version of the Munich Olfaction Test.This test is based on the sniff-bottle method and includes testsof (i) odor quality discrimination, (ii) intensity discrimination,(iii) detection thresholds, and (iv) recognition, hedonic evaluationand identification ability. The patients could be divided intotwo distinct groups differing significantly on all four subtestsand showing no overlap in performance: 20 anosmics, conformingto Kallmann's syndrome, and 17 apparent normosmics whose performancewas slightly poorer, but not significantly different to thatof the controls. The unexpected failure to find a continuumof olfactory dysfunction now raises the question whether HHwith or without anosmia represents two syndromes with distinctetiologies, or rather reflects the ability of the olfactorysystem to function well despite morphological impairment.     

Bone remodelling induced by physical stress is prostaglandin E2 mediated

In vitro cultured bone cells were found to be responsive to hormones and physical forces. A simple device has been developed which enables the direct application of physical forces to tissue culture dishes to which cells are firmly attached. The physical forces created a deformation of the dish. It was found that prostaglandin E₂ synthesis underwent a rapid increase, reaching a maximum after 20 min and then declined. Concurrent with the increase in prostaglandin E₂ was an increase in cyclic AMP production, having a maximum around 15 min. The increase in cyclic AMP was blocked by indomethacin, the prostaglandin E₂ synthesis inhibitor, indicating the dependence of cyclic AMP production on the de novo synthesis of prostaglandin E₂. Prostaglandin E₂ added to cells mimicked the effect of physical forces on the production of cyclic AMP. The increase in cyclic AMP resulted from an early rise in adenyl cyclase activity (within 5 min) and a later (10 min) increase in phosphodiesterase activity. The same physical forces also stimulatedthe incorporation of thymidine into DNA after 24 h. On addition of prostaglandin E₂ the increase in DNA synthesis was also mimicked. Pretreatment of the cells with indomethacin abolished the effect of physical forces on DNA synthesis.The results suggest a stimulus receptor mechanism for physical forces which, like hormonal effectors, are mediated by prostaglandins and stimulate cyclic AMP and DNA synthesis.We believe that physical forces stimulate bone remodelling through such a stimulus receptor system, mediated by prostaglandins.     

Direct mapping of rare messenger RNAs by means of oligomer-directed ribonuclease H cleavage

A method has been developed for characterizing rare messenger RNAs in the bulk population by using oligodeoxyribonucleotide: RNA hybrids as substrates for Escherichia coli ribonuclease H. Two 1.3-kb mRNAs in lymphocyte cytoplasm, interferon-gamma (0.002% of polyadenylated mRNA), and prothymosin-alpha, have been studied. Interferon-gamma mRNA was cut virtually completely into two fragments, each about 0.6 kb in length, by using an interferon-specific 24-mer to direct cleavage. Prothymosin-alpha mRNA in the same bulk population was unaffected by this treatment. When the 24-mer was replaced by a 12-mer, whose sequence was based on an incomplete cDNA clone for prothymosin-alpha, the products included two fragments of prothymosin-alpha mRNA. The sum of the fragment lengths equaled the length of the mRNA. Although the reaction directed by the smaller oligomer did not go to completion, the 12-mer, and hence the cDNA clone from which it was derived, could nevertheless be oriented with respect to prothymosin-alpha mRNA. With this technique, sequences in mRNA can be mapped without first isolating full-length cDNA clones.     

Photosensitized formation of 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) in DNA by riboflavin.

Potosensitized formation of 8-hydroxyguanine in DNA by riboflavin was observed. A reaction mechanism involving guanine radical cation and hydration reaction was proposed. This hypothesis was confirmed by the incorporation of [18O]-atom within guanine moiety in isotopic experiments using [18O]-H2O. Photosensitized formation of oh8Gua by riboflavin was also observed in cellular DNA.