[Show abstract][Hide abstract] ABSTRACT: We previously reported that a pool of low molecular weight peptides can be extracted by alkali treatment of DNA preparations obtained from prokaryotic and eukaryotic cells after intensive deproteinization. This class of peptides, isolated from wheat bud chromatin, induces growth inhibition, DNA damage, G2 checkpoint activation and apoptosis in HeLa cells. In this work we studied their mechanism of action by investigating their ability to interfere with DNA synthesis.
BrdUrd comet assays were used to detect DNA replication defects during S phase. DNA synthesis, cell proliferation, cell cycle progression and DNA damage response pathway activation were assessed using 3H-thymidine incorporation, DNA flow cytometry and Western blotting, respectively.
BrdUrd labelling close to DNA strand discontinuities (comet tails) detects the number of active replicons. This number was significantly higher in treated cells (compared to controls) from entry until mid S phase, but markedly lower in late S phase, indicating the occurrence of defective DNA synthesis. In mid S phase the treated cells showed less 3H-thymidine incorporation with respect to the controls, which supports an early arrest of DNA synthesis. DNA damage response activation was also shown in both p53-defective HeLa cells and p53-proficient U2OS cells by the detection of the phosphorylated form of H2AX after peptide treatment. These events were accompanied in both cell lines by an increase in p21 levels and, in U2OS cells, of phospho-p53 (Ser15) levels. At 24 h of recovery after peptide treatment the cell cycle phase distribution was similar to that seen in controls and CDK1 kinase accumulation was not detected.
The data reported here show that the antiproliferative effect exhibited by these chromatin peptides results from their ability to induce genomic stress during DNA synthesis. This effect seems to be S-phase specific since surviving cells are able to progress through their normal cell cycle when the peptide fraction is removed from the culture medium. It is likely that the subsequent apoptosis is a consequence of the failed attempt of the tumour cells to repair the DNA damage induced by the peptides.
[Show abstract][Hide abstract] ABSTRACT: In insect renal physiology, cGMP and cAMP have important regulatory roles. In Drosophila melanogaster, considered a good model for molecular physiology studies, and in other insects, cGMP and cAMP act as signalling molecules in the Malpighian tubules (MTs). However, many questions related to cyclic nucleotide functions are unsolved in principal cells (PC) and stellate cells (SC), the two cell types that compose the MT. In PC, despite the large body of information available on soluble guanylate cyclase (sGC) in the cGMP pathway, the functional circuit of particulate guanylate cyclase (pGC) remains obscure. In SC, on the other side, the synthesis and physiological role of the cGMP are still unknown. Our biochemical data regarding the presence of cyclic nucleotides in the MTs of Rhyacophila dorsalis acutidens revealed a cGMP level above the 50%, in comparison with the cAMP. The specific activity values for the membrane-bound guanylate cyclase were also recorded, implying that, besides the sGC, pGC is a physiologically relevant source of cGMP in MTs. Cytochemical studies showed ultrastructurally that there was a great deal of pGC on the basolateral membranes of both the principal and stellate cells. In addition, pGC was also detected in the contact zone between the two cell types and in the apical microvillar region of the stellate cells bordering the tubule lumen. The pGC signal is so well represented in PC and, unexpectedly in SC of MTs, that it is possible to hypothesize the existence of still uncharacterized physiological processes regulated by the pGC-cGMP system.
[Show abstract][Hide abstract] ABSTRACT: The ultracytochemical localization of particulate guanylate cyclase (GC) has been studied in Rana esculenta choroid plexus after activation with rat atrial natriuretic peptide (ANP), porcine brain natriuretic peptide (BNP) and porcine C-type natriuretic peptide (CNP). This study shows that the three peptides are activators of GC in the choroid plexus as demonstrated by the presence of reaction products at the level of the epithelium and sinusoids. In the apical zone of the epithelial cells predominantly BNP seems to activate GC, while ANP and CNP activate GC mainly at the level of the lateral surfaces. Moreover, ANP stimulates the enzyme along the basal membrane of the epithelial cells as well as the membranes of the endothelium of the sinusoids. In the presence of CNP, enzyme stimulation can also be found at the level of the endocellular membranes. These results confirm that the choroid plexus is an organ with receptors for the natriuretic peptides which are involved in the processes of osmoregulation and the control of cerebrospinal fluid production.
Journal of submicroscopic cytology and pathology 08/1998; 30(3):355-63.
[Show abstract][Hide abstract] ABSTRACT: This study shows that in the choroid plexus of Rana esculenta particulate guanylate cyclase (GC) is appreciably stimulated by porcine brain natriuretic peptide (BNP). Ultracytochemical tests for GC show that BNP notably increases the enzymatic reaction product along the apical surfaces of the epithelial cells. It can therefore be hypothesized that the apical zone of the epithelial cells possess receptors which have a particular affinity for BNP produced in the central nervous system and dumped into the cerebrospinal fluid. These results, together with those of a previous study , confirm that the choroid plexus is an organ which has receptors for the natriuretic peptides which are involved in the processes of osmoregulation and the control of cerebrospinal fluid production.
Brain Research 01/1996; 705(1-2):295-301. DOI:10.1016/0006-8993(95)01178-1 · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study shows that the choroid plexus of Rana esculenta contains a guanylate cyclase particulate (GCp), similar to that identified in Mammalia, that is quite sensitive to the atrial natriuretic factor (ANF). The cytochemical tests for GCp show that ANF increases the enzymatic reaction products. Deposits are observed on the apical portion, at the basal level and along the lateral edges of the epithelial cells, with the exclusion of some intercalary epithelial cells with reaction-lacking microvilli. In particular, ANF seems to intensely stimulate the GCp activity along the lateral membranes of the epithelial cells delimiting enlarged intercellular spaces, which are probably dilated for the transport of water and solutes. These data confirm the osmoregulatory role of the hormone and its control of cephalorachidian liquid composition.
Tissue and Cell 05/1995; 27(2):233-40. DOI:10.1016/S0040-8166(95)80025-5 · 1.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 1. During the first stages of embryonic development of Bufo bufo, the levels of cAMP and cGMP showed interesting opposite trends analogous to the trends of their respective enzymes. 2. In the late segmentation, the high increase of guanylate cyclase specific activity and the corresponding rise of embryonic level of cGMP, could indicate the involvement of this nucleotide in cellular proliferation. 3. During the following stage of the gastrulation, the increase of adenylate cyclase specific activity, coupled to the loss of guanylate cyclase specific activity, could suggest the importance of cAMP in the phenomena of differentiation induction. 4. Furthermore, the cytochemical investigation of adenylate and guanylate cyclase localization seems to confirm the prominent role of cAMP during the differentiation phases.
Comparative Biochemistry and Physiology Part A Physiology 07/1993; 105(2):319-22. DOI:10.1016/0300-9629(93)90214-O · 2.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 1. The presence of high-Mr and low-Mr acid phosphatases [orthophosphoric-monoester phosphohydrolase, (acid optimum), EC 184.108.40.206] in the skeletal muscle of frog Rana esculenta was reported. 2. The subcellular localization and some characteristics of both enzymes were also described. 3. The low-Mr AcPase was purified to homogeneity. The enzyme did not absorb on Concanavalin A-Sepharose 4B indicating that this was not a glycoprotein. 4. The enzyme is homogeneous on polyacrylamide gel electrophoresis and moves as a single band of Mr 13.7 +/- 0.8 kDa in the presence of sodium dodecyl sulphate. 5. The Mr of the native enzyme was 14.0 +/- 1.1 kDa as determined by gel filtration on a Sephadex G-100 column. The isoelectric point was 6.02. 6. The enzyme was strongly inhibited by 1 mM Ag+, Hg2+, Sn2+ and Cu2+ while other cations both at 10(-2) and 10(-3) M showed little or no effect. 7. The enzyme was insensitive to NaF and tartrate but was strongly deactivated by formaldehyde, PMB, Iodoacetamide and Triton X-100. Phosphate was a competitive inhibitor (k1 = 0.83 mM). 8. The best substrate for the enzyme was p-nitrophenylphosphate but phenylphosphate, flavin mononucleotide and o-P-tyrosine were also hydrolyzed, though at different rates. 9. The enzyme activity was enhanced in the presence of methanol, ethanol, acetone and glycerol indicating a phosphotransferase activity.
International Journal of Biochemistry 02/1991; 23(10):1115-22. DOI:10.1016/0020-711X(91)90152-D · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing] has been cytochemically evidentiated in the cells which make-up the lung air-blood barrier. The cytochemical procedure utilized demonstrates the presence of membrane-bound guanylate cyclase activity through precipitation of lead pyrophosphate in tissues incubated with GTP or with guanylyl imidodiphosphate. Electron microscopic examination reveals that guanylate cyclase (GC) is localized, as micropinocytic vesicles, within endothelial components of small blood vessels, in basal lamina and in the flat alveolar cells. The secretory alveolar cells also exhibit the positive GC reactivity in their peripheric cytoplasm and in their microvilli. The observations support that GC and cGMP are involved in cellular transport phenomena. The enzyme might play a role in the secretion process of surface active material. Positive staining has been found also in other types of cells, namely alveolar macrophages and fibroblasts. A biochemical evaluation of GC activity shows that about 30-40% of this activity is associated with the particulate fraction, which justifies its abundance in the cytochemical reports shown in the paper.
Tissue and Cell 02/1991; 23(1):67-74. DOI:10.1016/0040-8166(91)90067-4 · 1.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study was conducted to determine the possible correlations between cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP), and haemoglobin (Hb) concentration in nucleated cell suspensions of rabbit bone marrow incubated with erythropoietin (Ep). The levels of cAMP and cGMP were measured following the addition of different Ep concentrations to the suspensions. The Hb concentration was also measured in suspensions treated with Ep, dibutyryl cAMP (db-cAMP) or dibutyryl cGMP (db-cGMP), respectively. The following results were obtained: (1) upon the addition of 1 IU ml-1 Ep, an increase of cAMP levels was related to an increase in Hb concentration; while a decrease of Hb concentration was related to an increase of cGMP levels obtained when 0.1 IU ml-1 Ep was present in the incubation mixture. (2) A mimetic effect on Hb concentration was obtained upon the addition of db-cAMP or db-cGMP to the suspensions. (3) A quantitative correlation was found between the cAMP/cGMP ratio and Hb levels in cellular suspensions. This rapport was reviewed with respect to the controls as a decrease in Hb concentration when the ratio is less than one and an increase in Hb concentration when the ratio is greater than one.
Cell Biochemistry and Function 04/1984; 2(2):119-24. DOI:10.1002/cbf.290020213 · 2.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Within the realm of the general hypothesis concerning the role of cGMP on intracellular calcium regulation in biological systems, we have investigated the action of cyclic nucleotides during excitation-contraction coupling in frog sartorius muscle. Our data show that several guanosine nucleotides (GTP, GDP, dibutyryl-cGMP) can increase the isometric twitch tension with a maximum increase of 40% in the muscles treated with cGMP. This increase is completely independent of external Ca2+ concentration. The use of dantrolene sodium (known to inhibit calcium release from sarcoplasmic reticulum) results in a decrease in the twitch tension with a contemporary decrease in the intracellular levels of cGMP; whereas, the addition of cGMP to the muscles treated with dantrolene antagonizes, at least partially, the effect of the drug on tension development. Finally, in chemically skinned muscles, cGMP induces a reversible contracture equal to approximately one-half of that evoked by 10(-4) M Ca2+.
Canadian Journal of Physiology and Pharmacology 07/1983; 61(6):590-4. DOI:10.1139/y83-090 · 1.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tetanic stimulation at different temperatures (5 and 20 °C) of the frog sartorius muscle results in an increase of cyclic guanosine monophosphate (cGMP) directly correlated to the tension developed. Cyclic adenosine monophosphate (cAMP) levels change differently for different temperature values. The variations could be explained by the interaction between Ca2+ and the enzymes which control cyclic nucleotide levels, namely, adenylate cyclase, guanylate cyclase, and phosphodiesterase.
Canadian Journal of Physiology and Pharmacology 02/1982; 60(1):79-83. DOI:10.1139/y82-011 · 1.77 Impact Factor