Acetic Acid-urea Research Articles

One-pot synthesis of 3,4-dihydropyrimidin-2(1 H)-ones using boric acid as catalyst

A simple effective synthesis of 3,4-dihydropyrimidin-2(1 H)-one derivatives, using boric acid as catalyst, from aromatic aldehydes, 1,3-dicarbonyl compounds and urea in glacial acetic acid is described. Compared with the classical Biginelli reaction conditions, this new method has the advantage of excellent yields (86–97%) and short reaction time (0.5–2 h).

Protein Kinase CK2 Phosphorylates the High Mobility Group Domain Protein SSRP1, Inducing the Recognition of UV-damaged DNA

The structure-specific recognition protein SSRP1 plays a role in transcription and replication in the chromatin context. Mediated by its C-terminal high mobility group (HMG) box domain, SSRP1 binds DNA non-sequence specifically but recognizes certain DNA structures. Using acetic acid urea polyacrylamide gel electrophoresis and mass spectrometry, we have examined the phosphorylation of maize SSRP1 by protein kinase CK2 alpha. The kinase phosphorylated several amino acid residues in the C-terminal part of the SSRP1 protein. Two phosphorylation sites were mapped in the very C-terminal region next to the HMG box domain, and about seven sites are localized within the acidic domain. Circular dichroism showed that the phosphorylation of the two C-terminal sites by CK2 alpha resulted in a structural change in the region of HMG box domain, because the negative peak of the CD spectrum at 222 nm was decreased by approximately 10%. In parallel, the phosphorylation induced the recognition of UV-damaged DNA, whereas the non-phosphorylated protein does not discriminate between UV-damaged DNA and control DNA. The affinity of CK2 alpha-phosphorylated SSRP1 for the DNA correlates with the degree of UV-induced DNA damage. Moreover, maize SSRP1 can restore the increased UV-sensitivity of a yeast strain lacking the NHP6A/B HMG domain proteins to levels of the control strain. Collectively, these findings indicate a role for SSRP1 in the UV response of eukaryotic cells.

Synthesis of Pyrimidin‐2‐ones and Imidazolin‐2‐ones.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Unprecedented reactivity of 3-amino-1 H,3 H-quinoline-2,4-diones with urea: an efficient synthesis of 2,6-dihydro-imidazo[1,5- c]quinazoline-3,5-diones

Substituted 3-amino-1 H,3 H-quinoline-2,4-diones react with urea in acetic acid to give novel 2,6-dihydro-imidazo[1,5- c]quinazoline-3,5-diones in high yields. The same compounds were obtained, albeit with small yields, from 3-chloro-1 H,3 H-quinoline-2,4-diones and urea. In the proposed reaction mechanism, a molecular rearrangement of the primarily formed mono-substituted urea takes place. The prepared 2,6-dihydro-imidazo[1,5- c]quinazoline-3,5-diones were characterized by their 1H, 13C, 15N NMR and IR spectra and atmospheric pressure chemical ionisation mass spectra.

Synthesis of pyrimidin-2-ones and imidazolin-2-ones

While reaction of chalcone dibromides (1) with urea in acetic acid is known to give 5-aryl-4-a-bromobenzylimidazolin-2-ones (2), that of 4-methoxychalcone dibromides afforded 4-aryl-5-arylmethineimidazolin-2-ones (4) and 4,6-diaryl-5-bromo-5,6-dihydropyrimidin-2(1H)-ones (5).

Histone H1 is phosphorylated in non-replicating and infective forms of Trypanosoma cruzi

The nuclear structure changes during the differentiation from growing to infective stages of Trypanosoma cruzi. As histone modifications have been correlated with structural and functional changes of chromatin, we investigated whether histones in T. cruzi are modified during the life cycle of this protozoan parasite. We found that histone H1 isolated from proliferating forms (epimastigotes) and from differentiated/infective forms (trypomastigotes) have a distinct migrating pattern in Triton–acetic acid–urea gel electrophoresis. While epimastigotes contain predominantly a fast migrating form, a slow migrating band is prominent in trypomastigotes. By metabolically labeling the cells with radioactive phosphate, we demonstrated that the slow migrating histone H1 band is phosphorylated, and that after alkaline phosphatase treatment, it migrates as the fast form. Parasites arrested at the onset of the S phase of the cell cycle with hydroxyurea (HU) also predominantly have the phosphorylated form of histone H1, suggesting that phosphorylation occurs in non-replicating stages of T. cruzi. We also found that the phosphorylated histone H1 is more weakly associated with the chromatin, being preferentially released at 150 mM NaCl. Therefore, histone H1 phosphorylation varies during the life cycle of T. cruzi, and might be related to changes in the chromatin structure.

Butyrate upregulates stromelysin-1 production by intestinal mesenchymal cells

Nutritional factors and resident bacteria participate in the pathogenesis of intestinal inflammation. However, the ways in which bacteria and complex diets might modulate matrix metalloproteinase (MMP) production are unknown. We hypothesized that butyrate might enhance production of MMPs, thus amplifying their response to signals in inflammatory conditions. Human mesenchymal cells were incubated with butyrate and then stimulated with cytokines. MMPs and inhibitors were studied by Western blotting and quantitative RT-PCR. Acetylation of histones was examined in Triton X acetic acid-urea gels by PAGE. We showed that butyrate selectively enhanced the protein production and mRNA expression of stromelysin-1 in tumor necrosis factor-alpha- or interleukin-1beta-stimulated mesenchymal cells. Butyrate alone did not induce any change in MMP production or mRNA expression. It increased the acetylation of histones in mesenchymal cells. Furthermore, acetylation of histones (induced by trichostatin A) reproduced the effects of butyrate. Although butyrate is a major source of nutrient for the colonic epithelial cells, it modulates intestinal inflammation through the secretion of stromelysin-1 in stimulated stromal cells via the inhibition of histone deacetylase.

Separation of Histone Variants and Post‐Translationally Modified Isoforms by Triton/Acetic Acid/Urea Polyacrylamide Gel Electrophoresis

Due to their similarities in size and charge, complete resolution of histones by electrophoresis poses a considerable challenge. The addition of nonionic detergents to the traditional acetic acid/urea (AU) polyacrylamide gel electrophoresis (PAGE) system has afforded an excellent method to separate not only the different modified forms of histones, but also the primary sequence variant subtypes of selected histone species; it is widely used to separate histones with varying levels of acetylation. This unit describes the use of gels containing the nonionic detergent Triton X-100, referred to as Triton/acetic acid/urea (TAU) polyacrylamide gels, for analysis of histones. Also included are support protocols detailing several accessory techniques: assembly of gel plates for the TAU gel, preparation of histones from isolated nuclei in a solubilized form amenable to electrophoresis, and electrophoretic transfer of proteins from these gels to PVDF membranes.

Isolation and preliminary characterization of histone H1.b allelic variants from quail erythrocytes

Our goal was to purify and characterize the allelic variants H1b1 and H1b2 of histone H1.b, one of the seven subtypes of this linker histone extracted from Japanese quail erythrocyte nuclei. These variants are revealed phenotypically as band H1.3 or part of band H1.4 by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). All H1 subtypes together were separated from H5 by gel-permeation chromatography through Bio-Gel P-150. H1 was then fractionated on a column of the cation-exchange resin Amberlite CG-50 by using a shallow guanidine hydrochloride gradient, which enriched subtype H1.b together with H1.z and overlapping with subtypes H1.a and H1.b. Alternatively purification of subtypes was achieved electrophoretically: total H1 fractions from quail with different H1 phenotypes were first resolved into sub-types by PAGE in acetic acid-urea; after staining, the appropriate H1.b bands from several parallel gel pieces were excised and the histone was concentrated by PAGE in SDS. After fragmentation of H1.b in the gel pieces with N-bromosuccinimide (NBS), PAGE in SDS indicated no difference between H1b1 and H1b2 in the C-terminal "half" of the polypeptides. In contrast, limited digestion with endoprotease V8 from Staphylococcus aureus has shown that differences, probably by a few residues in length, reside in the N-terminal part of the molecule, close to the amino-terminus.

Macrophage inflammatory protein-2: chromosomal regulation in rat small intestinal epithelial cells.

Nonpathogenic, resident bacteria participate in the pathogenesis of inflammation in the small intestine, but the molecular messages produced by such bacteria are unknown. Inflammatory responses involve the recruitment of specific leukocyte subsets. We, therefore, hypothesized that butyrate, a normal bacterial metabolite, may modulate chemokine secretion by epithelial cells, by amplifying their response to proinflammatory signals. We studied the expression of the chemokine, macrophage inflammatory protein-2 (MIP-2) by the rat small intestinal epithelial cell line, IEC-6. Cells were stimulated with lipopolysaccharide or with interleukin 1beta (IL-1beta) and incubated with sodium butyrate. Acetylation of histones was examined in Triton X acetic acid-urea gels by PAGE. Unstimulated IEC-6 cells did not secrete MIP-2. However, lipopolysaccharide and IL-1beta induced MIP-2 expression. Butyrate enhanced MIP-2 secretion both in lipopolysaccharide-stimulated and IL-1beta-stimulated enterocytes; but butyrate alone did not induce MIP-2 expression. Butyrate increased the acetylation of histones extracted from the nuclei of IEC-6 cells. Furthermore, acetylation of histones (induced by trichostatin A, a specific inhibitor of histone deacetylase) enhanced MIP-2 expression by cells stimulated with IL-1beta. In conclusion, trichostatin A reproduced the effects of butyrate on MIP-2 secretion. Butyrate, therefore, increases MIP-2 secretion in stimulated cells by increasing histone acetylation. We speculate that butyrate carries information from bacteria to epithelial cells. Epithelial cells transduce this signal through histone deacetylase, modulating the secretion of chemokines.

Isolation of histones and related chromatin structures from spermatozoa nuclei of a dasyurid marsupial, Sminthopsis crassicaudata

The spermatozoa of a dasyurid marsupial, Sminthopsis crassicaudata, have two distinct nuclear regions: uniformly electron-dense chromatin (C1) in the interior and fissured chromatin (C2) at the periphery. To investigate whether the differences in morphology are due to incorporation of different packaging proteins, spermatozoa nuclear proteins were characterised by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) and fractionated by reverse-phase high-pressure liquid chromatography (HPLC). The main protein component was protamine I, but a complete histone complement (H1, H2A, H2B, H3, and H4) was also detected. Immunocytochemistry showed localisation of H4, H2B, and H2A histones to the periphery of the nuclei, a region that corresponded to the C2 chromatin. The fissures in the chromatin of this region disappeared following incubation with fish protamines, indicating that the nucleohistone C2 region may be incompletely condensed relative to nucleoprotamines. This observation is consistent with the view that 60% of phosphodiester charges remain negative in nucleohistone DNA, whereas all DNA charges are neutralised in highly compact nucleoprotamines. Treatment of spermatozoa with micrococcal nuclease showed that the C1 chromatin was resistant to digestion, whereas the C2 region was cleaved into 30- to 38-nm agglomerates and 11-nm nucleosomal-size structures. Thus, this study demonstrates that spermatozoa nuclei of this marsupial species contain peripherally localised histones, and the nucleohistone chromatin accounts for the different morphology of the C2 region compared with the rest of the nucleus. J. Exp. Zool. 278:322–332, 1997. © 1997 Wiley-Liss, Inc.

Isolation of histones and related chromatin structures from spermatozoa nuclei of a dasyurid marsupial,Sminthopsis crassicaudata

The spermatozoa of a dasyurid marsupial, Sminthopsis crassicaudata, have two distinct nuclear regions: uniformly electron-dense chromatin (C1) in the interior and fissured chromatin (C2) at the periphery. To investigate whether the differences in morphology are due to incorporation of different packaging proteins, spermatozoa nuclear proteins were characterised by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) and fractionated by reverse-phase high-pressure liquid chromatography (HPLC). The main protein component was protamine I, but a complete histone complement (H1, H2A, H2B, H3, and H4) was also detected. Immunocytochemistry showed localisation of H4, H2B, and H2A histones to the periphery of the nuclei, a region that corresponded to the C2 chromatin. The fissures in the chromatin of this region disappeared following incubation with fish protamines, indicating that the nucleohistone C2 region may be incompletely condensed relative to nucleoprotamines. This observation is consistent with the view that 60% of phosphodiester charges remain negative in nucleohistone DNA, whereas all DNA charges are neutralised in highly compact nucleoprotamines. Treatment of spermatozoa with micrococcal nuclease showed that the C1 chromatin was resistant to digestion, whereas the C2 region was cleaved into 30- to 38-nm agglomerates and 11-nm nucleosomal-size structures. Thus, this study demonstrates that spermatozoa nuclei of this marsupial species contain peripherally localised histones, and the nucleohistone chromatin accounts for the different morphology of the C2 region compared with the rest of the nucleus.

Histone H1 and Core Histones inLeishmaniaandCrithidia:Comparison withTrypanosoma

The Trypanosomatidae family is characterized by flagellated protozoa presenting a kinetoplast. Several genera of this family contain species that are pathogenic to man and domestic animals. Their chromatin is not condensed into chromosomes during cell division. As a contribution to the understanding of basic aspects of their genome organization, we present a systematic characterization of the histones from three genera of the Trypanosomatidae family.Crithidia fasciculataandLeishmania mexicanashow core nucleosomal histones with electrophoretic mobilities both similar to and different from those ofTrypanosoma cruziand higher eukaryotes. Another protein is extracted from the chromatin of these organisms by procedures designed to purify histone H1. This protein presents elution profiles by HPLC and amino acid composition of histone H1. Considering these data and the high mobility of this protein in Triton–acetic acid–urea–polyacrylamide gel electrophoresis, as well as its position relative to the nucleosomal core histones in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, we postulate thatCrithidiaandLeishmaniapossess a histone H1 shorter than that of higher eukaryotes as we have previously shown to be the case forT. cruzi.The possible presence of a shorter histone H1 in these trypanosomatids may explain the absence of chromatin condensation during cell division in these flagellates.

Novel 3,4-Di- and 3,4,5-Trihydroxyphenylalanine-Containing Polypeptides from the Blood Cells of the AscidiansAscidia ceratodesandMolgula manhattensis

Acetic acid urea extraction of the blood cells of two ascidian species yielded four novel families of polypeptides (3500–5300 Da) containing 3,4-dihydroxyphenylalanine (DOPA) and 3,4,5-trihydroxyphenylalanine (TOPA) from the PhlebobranchAscidia ceratodesand two DOPA proteins (9–10 kDa) from the StolidobranchMolgula manhattensis.3,4,5-Trihydroxyphenylalanine residues have not been found previously in polypeptides in any biological system. The DOPA content of theM. manhattensisproteins is the highest yet reported for a naturally occurring DOPA protein. The successful isolation of proteinaceous components fromA. ceratodesblood cells requires the incorporation of high concentrations of complexing agent in the extraction buffers to inactivate vanadium(III) which forms intractable organometallic polymers. TheA. ceratodesblood cell polypeptides are all rich in alanine and TOPA residues, have neutral to basic pIvalues, and, while all give single bands on acid urea–polyacrylamide electrophoresis, they exhibit extensive microheterogeneity. This is reflected by their large molecular weight distribution as determined by matrix-assisted laser desorption ionization–mass spectrometry, the variation in the ratio of their 280-nm absorption to chromophores of unknown structure in the polypeptide's electronic absorption spectra, and the N-terminal sequence analysis. The twoM. manhattensisproteins consist largely of four amino acids (alanine, valine, phenylalanine, and DOPA) with DOPA accounting for upward of 40 mol%. Both give an N-terminus of Ala-Phe-Tyr before resisting further progress by Edman degradation. Proteins and polypeptides from both ascidians are extremely resistant to proteases, a property which, while hampering characterization by sequence analysis, appears ideally suited to their proposed function of forming an impervious hemostat at the site of vascular injury. The yield of proteins and polypeptides relative to tunichromes appears to be seasonally dependent. The presence of DOPA and TOPA moieties in the proteins and polypeptides implicates them, as well as the tunichromes, as potential metal-sequestering agents.

Patterns of sperm-specific histone variation in sea stars and sea urchins: primary structural homologies in the N-terminal region of spermatogenic H1.

An electrophoretic characterization of histones from pyloric caeca, testes, and sperm of Asterias vulgaris revealed a sperm/testes-specific variant of histone H1 significantly larger than its somatic counterpart from pyloric caeca. Additional proteins were observed in H1 regions of acetic acid-urea polyacrylamide gels in testicular extracts. Sperm or testis-specific variants of H2B observed in sea urchins were not found in the sea star. Evidence presented suggests that sperm- or testes-specific H1 species of intermediate mobility may arise from a single, slow-migrating H1 species (SpH1). Although an increase in nonspecific DNA binding by nuclear proteins must occur during the process of spermatogenesis, different organisms exhibit various patterns of sperm-specific protein mediating differential binding during the process. Sperm-specific variants of both H1 and H2B histones are observed in sea urchins, while the only variant observed in sea stars during spermatogenesis is SpH1. Sequencing of the N-terminus of SpH1 from A. vulgaris revealed a repeating tetrapeptide in residues 3-6 and 8-11 (Ser-Pro-Arg-Lys and Ser-Pro-Lys-Lys, respectively), homologous to repeats in the N-termini of sperm-specific H1s from sea urchins. Primary structure within critical, variable regions of molecules responsible for nonspecific DNA binding appear conserved in many organisms. The occurrence of repeating tetrapeptides in SpH1 and other DNA binding proteins suggests that such domains may function similarly in various chromatins undergoing regulated or reversible condensation.

In vitro poly(ADPribosylation) of estrogen-induced proteins from the oviduct of the lizard Podarcis s. sicula Raf.

Poly(ADPribosylation) of nuclear proteins has been investigated in the nuclei from growing oviducts of intact and estrogen-treated spayed females of the lizard Podarcis s. sicula Raf. Isolated nuclei were incubated with [14C] NAD and nuclear proteins extracted in 0.2 m H2SO4. Labeled acid-soluble proteins were analysed by reverse-phase high-performance liquid chromatography (HPLC) and acetic acid-urea gel electrophoresis. The results reported here indicate that the ADPribosylation reaction is involved in modifying besides histone H2b, tissue specific proteins (SNPs and LMG-O). Moreover, comparable results have been obtained from nuclei prepared from the fully active oviduct of intact animals and spayed lizards stimulated with 17 beta-estradiol. It is concluded that the poly-(ADPribosylation) of hormone-induced proteins might play a role in the differentiation of the lizard oviduct.

Analysis of age-associated alteration in the synthesis of HMG nonhistone proteins of the rat liver

HMG proteins were extracted with 5% PCA or 0.35 M NaCl from whole tissue, nuclei or chromatin of the liver of young (19 weeks) and old (118 weeks) male rats. They were resolved on acetic acid-urea polyacrylamide gel. The electrophoretic patterns of the major HMG proteins 1, 2, 14 and 17 of both ages are similar. The in vitro synthesis of HMG 1 and 2 decreases, but that of HMG 14 and 17 increases considerably in the liver of old rats. The synthesis of different HMG proteins is modulated differentially by spermine, butyrate, dexamethasone and 3-aminobenzamide in the liver of young and old rats. These findings suggest that HMG proteins contribute to alterations in the organization of chromatin and expression of genes during aging.

A comparison of the histone H1 complements of avian erythrocytes

1. 1. Histone HI from chicken, turkey, duck and goose erythrocytes was resolved into six bands and that from quail into seven bands in an acetic acid-urea polyacrylamide gel. 2. 2. A fast migrating minor subtype H Le was detected in avian erythrocytes using two-dimensional polyacrylamide gel electrophoresis. 3. 3. Although histone subtype Hl.z from quail, turkey and duck was well separated in acid-urea gel, a similar protein in goose was found only in two-dimensional gel. This spot was absent in chicken. 4. 4. Histone HI spots .c, .c' and .d migrate in two-dimensional gel in a relatively constant manner forming a triangle-shaped pattern that facilitates comparison of HI subtypes among various avian species.

Nuclear proteins and chromatin structure in liver and intestinal epithelial cells of young growing and adult rats

Nuclei from liver and intestinal epithelial cells of young growing rats (39 days old) and adult rats (98 days old) were isolated. After addition of DNase I, the chromatin was separated by centrifugation (1100 g) into two fractions; the pellet (P) and the supernatant (S). The amount of chromatin released into the S-fraction was the same for the two age groups. The intestinal epithelial cell nuclei underwent self-digestion (in the absence of added DNase I) which was significantly higher in the young rats than in the adults. Subsequent examination using immunotechniques established the presence of non-sarcomeric myosin heavy-chain indicating that active genes were present for that protein. Hybridization of DNA with cDNA specific for myosin heavy-chain revealed that, relative to total DNA, the DNA retained in the P-fraction of both tissues and age groups contained the same amount of hybridizable sequences. In liver, nuclear proteins decreased significantly with age per g wet weight of tissue. In the enterocyte tissue, total DNA and protein increased with age. SDS-polyacrylamide gel or acetic acid-urea gel electrophoresis gave no age-related differences in the pattern of the proteins within each tissue. The results show that both liver nuclear DNA and protein decrease with age per g wet weight but increase per total tissue. In intestinal epithelial cells changes in chromatin structure with age were inherent within the nucleus.

ADP-ribosylation of nuclear proteins in rat ventral prostate during ageing

Poly(ADPR)polymerase activity and poly(ADP-ribosyl)ation of nuclear proteins have been investigated in ventral prostate nuclei of different aged rats (14, 28, 60, 180, 360 day old animals), by reverse-phase HPLC and acetic acid-urea polyacrylamide gel electrophoresis. The major ADP-ribose acceptor proteins were identified as histone H1 and H2b. It is concluded that concomitant with major changes to chromatin organization, poly(ADP-ribosyl)ation reaction is progressively inhibited during ageing of rat ventral prostate. These results support the hypothesis that prostatic dysfunction in senescent animals is related to a failure of DNA repair mechanisms and deregulated template activity.