Uridine Incorporation Research Articles

Inhibition of core histones acetylation by carcinogenic nickel(II)

Nickel, a well-established human carcinogen, was shown to decrease acetylation of histones H4 and H3 in cultured cells. Such a decrease is expected to suppress gene expression. However, nickel is known to not only suppress but also enhance the expression of many genes. So, perhaps, nickel can alter histone acetylation in a more complex way? In a first step of testing this presumption, we examined acetylation status of histones H2A, H2B, H3 and H4, in human (HAE) and rat (NRK) cells exposed to nickel(II) under various conditions. In both cell lines, acetylation of all four histones was down-regulated by nickel(II) in a concentration- and time-dependent manner. Acetylation of histone H2B was suppressed to greater extent than that of the others, with histone H3 being relatively least affected. The analysis of acetylation status of each of the four lysine sites at the N-terminal tail of histone H2B revealed decreases consistent with those observed in the total acetylation patterns, with the K12 and K20 residues being markedly more affected than K5 and K15 residues. Thus, the decrease in acetylation was to some degree site specific. In NRK cells, the observed uniform down-regulation of histone acetylation was consistent with a marked suppression of global gene transcription measured as [3H]-uridine incorporation into mRNA. However, in HAE cells, global RNA expression was transiently increased (in 24 h) before dropping below control after longer exposure (3 days). In conclusion, the effects of Ni(II) on histone acetylation are inhibitory, with their extent depending on the dose and exposure time. This uniform inhibition, however, is not consistently reflected in global RNA expression that in HAE cells may include both increase and decrease of the expression, clearly indicating the involvement of factors other than histone acetylation. The observed effects may contribute to neoplastic transformation of Ni(II)-exposed cells.

Abnormalities in Uridine Homeostatic Regulation and Pyrimidine Nucleotide Metabolism as a Consequence of the Deletion of the Uridine Phosphorylase Gene

We report in the present study the critical role of uridine phosphorylase (UPase) in uridine homeostatic regulation and pyrimidine nucleotide metabolism, employing newly developed UPase-/- mice. Our data demonstrate that the abrogation of UPase activity led to greater than a 6-fold increase in uridine concentrations in plasma, a 5-6-fold increase in lung and gut, and a 2-3-fold increase in liver and kidney, as compared with wild type mice. Urine uridine levels increased 24-fold normal in UPase-/- mice. Uridine half-life and the plasma retention of pharmacological doses of uridine were significantly prolonged. Further, in these UPase-/- mice, abnormal uridine metabolism led to disorders of various nucleotide metabolisms. In the liver, gut, kidney, and lung of UPase-/- mice, total uridine ribonucleotide concentrations increased 2-3 times as compared with control mice. Cytidine ribonucleotides and adenosine and guanosine ribonucleotides also increased, although to a lesser extent, in these organs. Most significant deoxyribonucleotide changes were present in the gut and lung of UPase-/- mice. In these tissues, dTTP concentration increased more than 4-fold normal, and dCTP, dGTP, and dATP concentrations rose 1-2 times normal. In kidney, dTTP concentration increased 2-fold normal, and dCTP and dGTP concentrations rose less than 1-fold normal. In addition, the accumulated uridine in plasma and tissues efficiently reduced 5-fluorouracil host toxicity and altered the anesthetic effect of pentobarbital. These data indicate that UPase is a critical enzyme in the regulation of uridine homeostasis and pyrimidine nucleotide metabolism, and 5-fluorouracil activity.

The nucleolus and its modifications during oogenesis of Torpedo marmorata

The structural organisation of the nucleolar apparatus during oogenesis of the spotted ray Torpedo marmorata was investigated. The observations showed that unlike other cartilaginous fishes, in T. marmorata the nucleolar apparatus was always represented by one or two conspicuous nucleoli, whose organization significantly changed during oocyte development. In the smallest follicles (follicles <300 μm in diameter) the nucleolus was made up of granular and fibrillar components, and actively incorporated 3H uridine; later it becomes more and more electron‐dense so in follicles of 400 μm in diameter its components and 3H uridine incorporation were no longer evident. These results indicate that in T. marmorata the nucleolar apparatus significantly changes and undergoes a possible impairment in rRNA synthesis. After nucleolus inactivation, the synthesis of rRNA may be substained by granulosa.

Alterations in pyrimidine nucleotide metabolism as an early signal during the execution of programmed cell death in tobacco BY-2 cells.

Changes in pyrimidine metabolism were investigated during programmed cell death (PCD) of tobacco BY-2 cells, induced by a simultaneous increase in the endogenous levels of nitric oxide (NO) and hydrogen peroxide. The de novo synthesis of pyrimidine nucleotides was estimated by following the metabolic fate of the (14)C-labelled orotic acid, whereas the rates of salvage and degradation pathways were studied by measuring the respective incorporation of (14)C-labelled uridine and uracil under different treatments. Nucleic acid metabolism was also examined using labelled thymidine as a marker. The results show that specific alterations in the balance of pyrimidine nucleotide synthesis, which include a decreased rate of salvage activity of uracil and uridine and increased salvage activity of thymidine, represent a metabolic switch that establishes proper cellular conditions for the induction of PCD. In particular, a reduction in the utilization of uracil for salvage products occurs very early during PCD, before the appearance of typical cytological features of the death programme, thus representing an early metabolic marker for PCD. These changes are strictly associated with PCD, since they do not occur if NO or hydrogen peroxide are increased individually, or if actinomycin, which inhibits the death programme, is added into the medium in the presence of NO and hydrogen peroxide. The possible roles of these fluctuations in pyrimidine metabolism on the cellular nucleotide pool are discussed in relation to the induction of cell death.

Synergistic effects of mupirocin and an isoflavanone isolated from Erythrina variegata on growth and recovery of methicillin-resistant Staphylococcus aureus

The phytochemical 2′,4′-dihydroxy-8-γ,γ-dimethylallyl-2″,2″-dimethylpyrano[5″,6″:6,7]isoflavanone (bidwillon B) was isolated from Erythrina variegata and its antibacterial properties against methicillin-resistant Staphylococcus aureus (MRSA) were investigated. Bidwillon B inhibited the growth of 12 MRSA strains at minimum inhibitory concentrations (MICs) of 3.13–6.25 mg/l, while MICs of mupirocin were 0.20–3.13 mg/l. The minimum bactericidal concentration (MBC) for bidwillon B and mupirocin against MRSA were 6.25–25 mg/l (MBC 90: 12.5 mg/l) and 3.13–25 mg/l (MBC 90: 25 mg/l), respectively. When bidwillon B and mupirocin were combined, synergistic effects were observed for 11 strains of MRSA (fractional inhibitory concentration indices, 0.5-0.75). The MBCs of mupirocin in the presence of bidwillon B (3.13 mg/l) were reduced to 0.05–1.56 mg/l. Bidwillon B at MIC values strongly inhibited incorporation of radio-labelled thymidine, uridine, glucose and isoleucine into MRSA cells. Mupirocin showed lower inhibitory effects than bidwillon B on thymidine, uridine and glucose incorporation, but incorporation of isoleucine was completely blocked with this antibiotic. These results indicate that bidwillon B possesses sufficient anti-MRSA activity for inhibiting growth and recovery, and that the compound acts synergistically with mupirocin. The results also suggest that both compounds act on MRSA via different mechanisms. Bidwillon B may prove to be a potent phytotherapeutic and/or combination agent with mupirocin in the elimination of nasal and skin carriage of MRSA.

Inhibition of mammalian RNA synthesis by the cytoplasmic Ca2+ buffer BAPTA. Analyses of [3H]uridine incorporation and stress-dependent transcription.

To determine whether oscillations of cytoplasmic [Ca(2+)] might be involved in transcription regulated by the unfolded protein response (UPR), dermal fibroblasts were loaded with the widely used Ca(2+) buffer BAPTA, which is expected to dampen cytoplasmic [Ca(2+)] changes without affecting resting [Ca(2+)]. BAPTA inhibited UPR-dependent transcription of the GRP78/BiP and EDEM genes. However, BAPTA also blocked cytoplasmic stress-dependent (UPR-independent) transcription of the HSP70 gene. These results led to the unexpected demonstration that BAPTA was a general inhibitor of cellular RNA synthesis in dermal fibroblasts. BAPTA is delivered to the cytoplasm as the acetoxymethyl (AM) ester BAPTA/AM, but released AM groups, as well as formaldehyde generated from AM breakdown, were ruled out as causes of RNA synthesis inhibition. BAPTA inhibited RNA synthesis in all mammalian cell types tested except CHO-K1. GRP78/BiP RNA induction in CHO-K1 cells was not blocked by BAPTA. Thus, there does not appear to be a critical requirement for cytoplasmic [Ca(2+)] changes in CHO-K1 UPR-dependent transcription. However, general inhibition of RNA synthesis by the [Ca(2+)] buffer BAPTA was unanticipated. This might possibly reflect a fortuitous interaction of BAPTA with the RNA synthesis machinery or a requirement for [Ca(2+)] changes.

Nucleic acid synthesis in cancerous cells under the effect of gnidilatimonoein from Daphne mucronata

Cytotoxicity evaluation of gnidilatimonoein, the most active isolated diterpene ester from Daphne mucronata [Sadeghi H, Mianabadi M, Yazdanparast R, (2002) Journal of Tropical. Medicinal Plant1 3: 169–173], revealed the strong antiproliferative activity among several different human cancer cell lines (K562, CCRF-CEM, HL-60 and MOLT-4 leukemia cell lines, LNCaP-FGC-10 a prostate cancer cell line) and a mouse BALB/C fibrosarcoma cell line (WEHI-164). Using flow cytometry technique, it was found that treatment of the most responsive cells (K562) with gnidilatimonoein inhibited the progression of cells through G 1 phase by almost 15% compared to the untreated cells. The population of the treated cells in the S and G 2 phases also reduced by 8.3% and 5.4%, respectively. Based on the extent of [ 3H]-thymidine and [ 3H]-uridine incorporation into DNA and RNA, respectively, the major metabolic effects of gnidilatimonoein were found to be mainly on DNA and to a less extent on RNA synthesis. Additionally, the activity of inosine-5′-monophosphate dehydrogenase (IMPDH), under the effects of genidilatimonoein, was reduced in the treated cells by 44%. These data strongly suggest that the purine biosynthetic pathway is significantly affected by gnidilatimonoein.

Antibacterial properties of a new isoflavonoid from Erythrina poeppigiana against methicillin-resistant Staphylococcus aureus.

A new isoflavonoid, together with four known isoflavonoids, was isolated from the roots of Erythrina poeppigiana. The chemical structure was determined by extensive spectroscopic studies, and then its antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) was investigated. The new isoflavonoid was identified as 3,9-dihyroxy-10-gamma,gamma-dimethylallyl-6a,11a-dehydropterocarpan (compound 1). Compound 1 inhibited bacterial growth most potently of the five isolates, and had a minimum inhibitory concentration (MIC) of 125 microg/ml against thirteen MRSA strains. Inhibitory activity was based on bactericidal action and viable cell number reduced by approximately 1/10,000 after 4 h incubation with compound 1. Despite intense bactericidal action against MRSA, compound 1 never resulted in leakage of 260 nm-absorbing substances from bacterial cells. Compound 1 (12.5 microg/ml) completely inhibited incorporation of radio-labeled thymidine, uridine and leucine into MRSA cells. Although glucose incorporation was also markedly inhibited by the compound, the amount of glucose incorporated by bacterial cells increased gradually with incubation time. These findings suggest that compound 1 exhibits anti-MRSA activity by interfering with incorporation of metabolites and nutrients into bacterial cells or by affecting the nucleic acids of MRSA cells. Furthermore, this new compound could be a potent phytotherapeutic agent for treating MRSA infections.

Pyrimidine deoxyribonucleotide metabolism during maturation and germination of white spruce (Picea glauca) somatic embryos: metabolic fate of C‐labelled cytidine, deoxycytidine and thymidine

Pyrimidine deoxyribonucleotide metabolism was investigated during maturation and germination of white spruce somatic embryos by following the metabolic fate of [2‐14C]cytidine, [2‐14C]deoxycytidine and [2‐14C]thymidine. The de‐novo pathway of deoxyribonucleotides was estimated indirectly, by the ability of the tissue to incorporate cytidine into DNA after conversion to dCTP. The salvage pathway was estimated by the utilization of labelled cytidine, deoxycytidine and thymidine for synthesis of deoxyribonucleotides and nucleic acids. Utilization of cytidine for DNA synthesis, via the de novo pathway, was always lower than that observed for RNA throughout the course of the experiment. Incorporation of cytidine into RNA was found to occur either directly, after conversion to CTP, mediated by the enzymes cytidine kinase, nucleoside monophosphate kinase and nucleoside diphosphate kinase, or indirectly, after conversion to UTP via uridine and UMP. Active incorporation of uridine into RNA of white spruce‐cultured cells was demonstrated previously. Salvage of deoxycytidine and thymidine was operative in maturing and germinating white spruce somatic embryos, as label from both compounds was recovered in nucleotides and DNA. However, the utilization of these precursors by the cells was different. Salvage of deoxycytidine was always higher than that observed for thymidine, which was extensively catabolized to CO2 at all stages of embryo development.

Protein synthesis inhibitory activity in culture filtrates from new strains of Streptomyces isolated from Brazilian tropical soils.

To investigate the effect of the culture supernatants from three newly isolated Streptomyces strains, 221, 235 and 606 on eukaryotic cells. Cell lines were treated with the culture filtrates and assayed for protein synthesis by metabolic labelling, followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. RNA synthesis was investigated by [5-3H]uridine incorporation. The three culture filtrates presented a strong inhibitory activity, reducing total protein synthesis of different eukaryotic cell lines by more than 85%. No effect on cellular RNA synthesis was detected. The culture filtrates did not affect the growth of the prokaryotic cells tested. These new Streptomyces strains, recently isolated from Brazilian tropical soils, produce molecule(s) with inhibitory activity specific to eukaryote protein synthesis. Streptomyces strains 221, 235 and 606, probably representing new species, might produce new bioactive compound(s), and can be used as valuable tools to study the protein synthesis pathway in eukaryotes.

Histone H4 Acetylation Patterns During Seed Germination and Early Plant Development

Here we studied whether early development of Silene latifolia is accompanied with changes in acetylation of nucleosomal histones H4. Using acid-urea-triton polyacrylamide gel and immunoblotting with specific antisera the histone acetylation in relationship with transcriptional activity, measured by [14C]-uridine incorporation, was analysed in dry and germinating seeds, seedlings, and adult leaves. We show that quiescent and germinating seeds, until the root tip is released from testa, are characterised by an absence of transcriptional activity and by a low H4 acetylation level: only mono-acetylated isoforms were present. During the postgermination period and early plantlet development, a high increase of transcriptional activity started and a sharp H4 hyperacetylation, up to the penta-acetylated isoform, was detected. We conclude that epigenetic modification by nucleosomal histone deacetylation plays a role in maintenance of global genome silencing in quiescent seeds.

Prolactin, cortisol, and insulin regulation of nucleoside uptake into mouse mammary gland explants.

Nucleosides are essential components of milk that are used for the nourishment of newborns. Effects of the three primary lactogenic hormones, including prolactin (PRL), insulin (I), and cortisol (H), on nucleoside uptake and incorporation into cultured mammary tissues taken from 12- to 14-day pregnant mice were determined; most experiments focused on the regulation of uridine uptake. Insulin alone, as well as PRL in the presence of insulin and cortisol, was shown to stimulate uridine uptake and incorporation into RNA in mammary explants taken from 12- to 14-day pregnant mice. The PRL effects were expressed at concentrations of 25 ng/ml and above, which are physiological plasma concentrations. In the absence of sodium, uridine uptake and incorporation were diminished, suggesting the presence of a sodium-dependent uridine transporter. In kinetic studies the apparent Km for uridine uptake was calculated to be 312 microM, and the Vmax 2.90 micromol/hr/L cell water; PRL had no effect on the Km but increased the Vmax to 5.88 micromol/hr/L cell water. When assessing uridine uptake in the presence of the other nucleosides at 0.1 mM, only cytidine competed with uridine uptake. The fact that distribution ratios of greater than 15:1 were achieved with uridine indicates that uridine uptake may be via an active transporter. These studies show that PRL enhances uridine update in mammary tissues by stimulating the activity, and probably synthesis, of a sodium-dependent, active uridine and cytosine transporter.

Imbalance of peripheral B lymphocytes and NK cells in rheumatoid arthritis.

The study was focused on several cellular immune disorders correlated with the imbalance between peripheral blood B lymphocytes and NK cells in severe rheumatoid arthritis. By flow cytometry we calculated the proportions of T, T helper, T cytotoxic/suppressor, B lymphocytes and natural killer cells in peripheral blood. The mitogen-induced proliferation of peripheral lymphocytes was measured by tritium-labeled uridine incorporation. Experimental data highlight a connection between annomal values of the B to natural killer cells ratio and disorders of the peripheral mononuclear cells concentration. We also showed that the polyclonal proliferation capacity of peripheral lymphocytes in rheumatoid arthritis is solely related to the B to natural killer cells ratio or to the natural killer cells proportion. The study reveals a potential role of the imbalance between proportions of peripheral B lymphocytes and natural killer cells in the immune pathogenesis of rheumatoid arthritis, thus pointing out an interrelation between the adaptive and innate immune systems.

Synthesis and antiviral evaluation of a mutagenic and non-hydrogen bonding ribonucleoside analogue: 1-beta-D-Ribofuranosyl-3-nitropyrrole.

Synthetic small molecules that promote viral mutagenesis represent a promising new class of antiviral therapeutics. Ribavirin is a broad-spectrum antiviral nucleoside whose antiviral mechanism against RNA viruses likely reflects the ability of this compound to introduce mutations into the viral genome. The mutagenicity of ribavirin results from the incorporation of ribavirin triphosphate opposite both cytidine and uridine in viral RNA. In an effort to identify compounds with mutagenicity greater than that of ribavirin, we synthesized 1-beta-D-ribofuranosyl-3-nitropyrrole (3-NPN) and the corresponding triphosphate (3-NPNTP). These compounds constitute RNA analogues of the known DNA nucleoside 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole. The 3-nitropyrrole pseudobase has been shown to maintain the integrity of DNA duplexes when placed opposite any of the four nucleobases without requiring hydrogen bonding. X-ray crystallography revealed that 3-NPN is structurally similar to ribavirin, and both compounds are substrates for adenosine kinase, an enzyme critical for conversion to the corresponding triphosphate in cells. Whereas ribavirin exhibits antiviral activity against poliovirus in cell culture, 3-NPN lacks this activity. Evaluation of 3-NPNTP utilization by poliovirus RNA-dependent RNA polymerase (RdRP) revealed that 3-NPNTP was not accepted universally. Rather, incorporation was only observed opposite A and U in the template and at a rate 100-fold slower than the rate of incorporation of ribavirin triphosphate. This diminished rate of incorporation into viral RNA likely precludes 3-NPN from functioning as an antiviral agent. These results indicate that hydrogen bonding substituents are critical for efficient incorporation of ribonucleotides into RNA by viral RdRPs, thus providing important considerations for the design of improved mutagenic antiviral nucleosides.

Influence of polyhydroxysteroids on [Ca(2+)](i).

Recently, we have shown that two biologically active, disulfated polyhydroxysteroids from the Pacific brittle star Ophiopholis aculeata stimulate Ca(2+) influx into different cell types. In the present study, 45Ca(2+) and two fluorescent calcium probes, quin-2/AM and fura-2/AM, were employed to investigate the course and amplitude of calcium signals induced in different mouse cells using an radio-isotope, spectrofluorimetry, and microcytofluorimetry techniques. The cytotoxic and hemolytic effects were not observed for both steroids at the wide range of concentrations. Steroids did not influence [3H]-uridine incorporation in a variety of cells. The investigated steroids stimulated a rapid increase in cytosolic Ca(2+) content in Ehrlich mouse carcinoma cells, mouse spleen lymphocytes, and mouse peritoneal macrophages in the concentration range 1-100 microg/ml on a dose-dependent basis. Blockers of L-type calcium channels, such as verapamil, diltiazem, nifedipine (1 x 10(-7)M), and 1mM EGTA, inhibited this process and reduced the response of cells to steroid application. The stimulatory effect of steroids on human fibroblast proliferation and mouse macrophage lysosome activity was observed also. It is suggested that the investigated compounds may act as Ca(2+)-agonists and increase Ca(2+)-transport across cell membranes.

Sphingosine 1-phosphate stimulates rat mesangial cell proliferation from outside the cells.

Proliferation of mesangial cells (MCs) is the initial step in glomerulonephritis, and platelet-derived mediators have been shown to play a significant role in this proliferation. Sphingosine 1-phosphate (S1P), one of the sphingolipids, is abundantly stored in platelets and is released upon stimulation. We examined the effects of S1P and related sphingolipids on the cell fate of cultured MCs in order to elucidate potential roles of these lipid mediators in glomerulonephritis. Cell proliferation was evaluated by bromodeoxy uridine (BrdU) incorporation together with MTS assay. Apoptosis of MCs was evaluated by examining annexin V staining and typical morphological changes in nuclei. We also examined the metabolism of [(3)H]sphingosine in MCs in either the presence or absence of platelet-derived growth factor (PDGF). The expression of endothelial differentiation genes (edg), which are the cell surface receptors for S1P in MCs, was examined by RT-PCR. S1P, but not the other sphingolipids, stimulated MC proliferation. In contrast, dimethylsphingosine (DMS) induced apoptosis in the MCs. The amount of sphingosine (Sph) converted into S1P was small and was not affected by PDGF. This observation suggested that Sph kinase activity producing S1P from Sph was low in the MCs. Furthermore, expression of edg-1, -2 and -5 in MCs was confirmed by RT-PCR. Our observations suggest that S1P stimulates MC proliferation from outside the cells, and not as a second messenger for PDGF. The modulation of MC fate with sphingolipids may provide possible strategies for the treatment of glomerulonephritis.

Hepatitis C Virus RNA-dependent RNA Polymerase (NS5B) as a Mediator of the Antiviral Activity of Ribavirin

Ribavirin is administered in combination with interferon-alpha for treatment of hepatitis C virus (HCV) infection. Recently, we demonstrated that the antiviral activity of ribavirin can result from the ability of a viral RNA polymerase to utilize ribavirin triphosphate and to incorporate this nucleotide with reduced specificity, thereby mutagenizing the genome and decreasing the yield of infectious virus (Crotty, S., Maag, D., Arnold, J. J., Zhong, W., Lau, J. Y., Hong, Z., Andino, R., and Cameron, C. E. (2000) Nat. Med. 6, 1375-1379). In this study, we performed a quantitative analysis of a novel HCV RNA polymerase derivative that is capable of utilizing stably annealed primer-template substrates and exploited this derivative to evaluate whether lethal mutagenesis of the HCV genome is a possible mechanism for the anti-HCV activity of ribavirin. These studies demonstrate HCV RNA polymerase-catalyzed incorporation of ribavirin opposite cytidine and uridine. In addition, we demonstrate that templates containing ribavirin support CMP and UMP incorporation with equivalent efficiency. Surprisingly, templates containing ribavirin can also cause a significant block to RNA elongation. Together, these data suggest that ribavirin can exert a direct effect on HCV replication, which is mediated by the HCV RNA polymerase. We discuss the implications of this work on the development of nucleoside analogs for treatment of HCV infection.

Effect of lead and chromium on nucleic acid and protein synthesis during sperm-zona binding in mice

Lead and chromium are ubiquitous environmental pollutants and their effects on reproductive physiology as shown by animal experiments and human studies is well documented. The present study was conducted to ascertain the role of these metals on gamete physiology during the sperm-zona binding process, Superovulated ova and capacitated sperm from BALB/c mice were exposed to lead (0.0, 0.2, 0. 5, 1.0 and 2.0 μg/ml) or chromium (0.0, 5.0, 10.0 and 20.0 μg/ml) for 2 h in the culture medium. The sperm that became attached to the ova in the presence of these metals were scored. Synthesis of DNA, RNA and protein was assessed during this process by labelling the cells with [ 3H]thymidine (20 μCi/ml), [ 3H]uridine (50 μCi/ml) and [ 35S]methionine (10 μCi/ml) in the culture medium after exposure to the metals for 2 h. Our studies show a significant dose-dependent decrease in the number of sperm attaching to the ova in both exposed groups along with a decrease in the incorporation of radiolabelled thymidine, uridine and methionine. The results indicate that DNA, RNA and protein synthesis under sperm-zona binding conditions are affected by the presence of these two metals. The physiology of the gametes is altered resulting in a low frequency of sperm attachment to the ova, which in turn leads to a risk of reproductive failure.

Brome mosaic virus replicase proteins localize with the movement protein at infection-specific cytoplasmic inclusions in infected barley leaf cells.

Subcellular localization of the Brome mosaic virus replicase-related 1a and 2a proteins, and the 3a movement protein in infected barley leaves was examined by immunogold electron microscopy. The 1a and 2a proteins colocalized at infection-specific electron-dense cytoplasmic inclusions. The 3a protein was also detected in these inclusions. The inclusions were oval or amorphous, and contained electron-lucent regions. Electron microscopic autoradiography showed that the inclusions were sites of [3H]uridine incorporation, suggesting that they are sites of viral RNA synthesis.

Structure and Mechanism of Action of an Indolicidin Peptide Derivative with Improved Activity against Gram-positive Bacteria

Indolicidin, an antimicrobial peptide with a unique amino acid sequence (ILPWKWPWWPWRR-NH(2)) is found in bovine neutrophils. A derivative of indolicidin, CP10A, has alanine residues substituted for proline residues and has improved activity against Gram-positive organisms. Transmission electron microscopy of Staphylococcus aureus and Staphylococcus epidermidis treated with CP10A showed mesosome-like structures in the cytoplasm. The peptide at 2-fold the minimal inhibitory concentration did not show significant killing of S. aureus ISP67 (a histidine, uridine, and thymidine auxotroph) but did show an early effect on histidine and uridine incorporation and, later, an effect on thymidine incorporation. Upon interaction with liposomes, detergents, and lipoteichoic acid, CP10A was shown by circular dichroism spectroscopy to undergo a change in secondary structure. Fluorescence spectroscopy indicated that the tryptophan residues were located at the hydrophobic/hydrophilic interface of liposomes and detergent micelles and were inaccessible to the aqueous quencher KI. The three-dimensional structure of CP10A in the lipid mimetic dodecylphosphocholine was determined using two-dimensional NMR methods and was characterized as a short, amphipathic helical structure, whereas indolicidin was previously shown to have an extended structure. These studies have introduced a cationic peptide with a unique structure and an ability to interact with membranes and to affect intracellular synthesis of proteins, RNA, and DNA.