Incorporation Of Radioactive Precursors Research Articles

Biosynthesis of heme O in intraerythrocytic stages of Plasmodium falciparum and potential inhibitors of this pathway

A number of antimalarial drugs interfere with the electron transport chain and heme-related reactions; however, the biosynthesis of heme derivatives in Plasmodium parasites has not been fully elucidated. Here, we characterized the steps that lead to the farnesylation of heme. After the identification of a gene encoding heme O synthase, we identified heme O synthesis in blood stage parasites through the incorporation of radioactive precursors. The presence of heme O synthesis in intraerythrocytic stages of Plasmodium falciparum was confirmed by mass spectrometry. Inabenfide and uniconazole–P appeared to interfere in heme synthesis, accordingly, parasite growth was also affected by the addition of these drugs. We conclude that heme O synthesis occurs in blood stage-P. falciparum and this pathway could be a potential target for antimalarial drugs.

Development of Methionyl-tRNA Synthetase Inhibitors as Antibiotics for Gram-Positive Bacterial Infections.

Antibiotic-resistant bacteria are widespread and pose a growing threat to human health. New antibiotics acting by novel mechanisms of action are needed to address this challenge. The bacterial methionyl-tRNA synthetase (MetRS) enzyme is essential for protein synthesis, and the type found in Gram-positive bacteria is substantially different from its counterpart found in the mammalian cytoplasm. Both previously published and new selective inhibitors were shown to be highly active against Gram-positive bacteria with MICs of ≤1.3 μg/ml against Staphylococcus, Enterococcus, and Streptococcus strains. Incorporation of radioactive precursors demonstrated that the mechanism of activity was due to the inhibition of protein synthesis. Little activity against Gram-negative bacteria was observed, consistent with the fact that Gram-negative bacterial species contain a different type of MetRS enzyme. The ratio of the MIC to the minimum bactericidal concentration (MBC) was consistent with a bacteriostatic mechanism. The level of protein binding of the compounds was high (>95%), and this translated to a substantial increase in MICs when the compounds were tested in the presence of serum. Despite this, the compounds were very active when they were tested in a Staphylococcus aureus murine thigh infection model. Compounds 1717 and 2144, given by oral gavage, resulted in 3- to 4-log decreases in the bacterial load compared to that in vehicle-treated mice, which was comparable to the results observed with the comparator drugs, vancomycin and linezolid. In summary, the research describes MetRS inhibitors with oral bioavailability that represent a class of compounds acting by a novel mechanism with excellent potential for clinical development.

Analysis of Sphingolipid Synthesis and Transport by Metabolic Labeling of Cultured Cells with [³H]Serine.

Analysis of lipid biosynthesis by radioactive precursor incorporation provides information on metabolic rates and the identity of rate-limiting enzymes and transporters. The biosynthesis of sphingolipids in cultured cells is initiated in the endoplasmic reticulum (ER) by the formation of a sphingoid base from serine and palmitoyl-CoA. N-acylation of the sphingoid base produces ceramide, which is transported to the Golgi apparatus where phosphocholine or carbohydrate headgroups are added to form sphingomyelin (SM) and complex glycosphingolipids (GSLs), respectively. Herein is described a protocol to measure ceramide and SM biosynthesis in cultured cells based on [(3)H]serine incorporation at the first step in the pathway. The method can be used to assay the effect of pharmacological and genetic manipulations on ceramide synthesis and transport to the Golgi apparatus.

Repurposing celecoxib as a topical antimicrobial agent.

There is an urgent need for new antibiotics and alternative strategies to combat multidrug-resistant bacterial pathogens, which are a growing clinical issue. Repurposing existing approved drugs with known pharmacology and toxicology is an alternative strategy to accelerate antimicrobial research and development. In this study, we show that celecoxib, a marketed inhibitor of cyclooxygenase-2, exhibits broad-spectrum antimicrobial activity against Gram-positive pathogens from a variety of genera, including Staphylococcus, Streptococcus, Listeria, Bacillus, and Mycobacterium, but not against Gram-negative pathogens. However, celecoxib is active against all of the Gram-negative bacteria tested, including strains of, Acinetobacter, and Pseudomonas, when their intrinsic resistance is artificially compromised by outer membrane permeabilizing agents such as colistin. The effect of celecoxib on incorporation of radioactive precursors into macromolecules in Staphylococcus aureus was examined. The primary antimicrobial mechanism of action of celecoxib was the dose-dependent inhibition of RNA, DNA, and protein synthesis. Further, we demonstrate the in vivo efficacy of celecoxib in a methicillin-resistant S. aureus (MRSA) infected Caenorhabditis elegans whole animal model. Topical application of celecoxib (1 and 2%) significantly reduced the mean bacterial count in a mouse model of MRSA skin infection. Further, celecoxib decreased the levels of all inflammatory cytokines tested, including tumor necrosis factor-α, interleukin-6, interleukin-1 beta, and monocyte chemo attractant protein-1 in wounds caused by MRSA infection. Celecoxib also exhibited synergy with many conventional antimicrobials when tested against four clinical isolates of S. aureus. Collectively, these results demonstrate that celecoxib alone, or in combination with traditional antimicrobials, has a potential to use as a topical drug for the treatment of bacterial skin infections.

Biosynthesis, targeting, and processing of lysosomal proteins: pulse-chase labeling and immune precipitation.

Incorporation of radioactive precursors of amino acids and/or modifier groups into proteins, isolation and sizing of polypeptide species of interest, and finally their detection and characterization provide a robust handle to examine the life cycle and varied modifications of any protein. A prerequisite in application of these techniques to lysosomal enzymes is the availability of an avid and specific antibody, because lysosomal proteins represent a very minor fraction of the cellular protein and must be purified without a significant loss many 1000-fold as conveniently as possible. Pulse-chase labeling and good knowledge on organelle-specific modifications of lysosomal proteins may enhance the information that can be obtained from such experiments. We describe procedures for pulse-chase labeling experiments that have proven to work with a commercially available antibody against a mouse and a human lysosomal protease and can be used as a reference in establishing the technique in any laboratory that has an access to a certified isotope facility and the knowledge to handle radioactivity safely. We discuss the crucial steps and refer to alternatives described in the literature. The present model protein cathepsin Z is synthesized as a larger proenzyme that contains two N-linked oligosaccharides and matures to a shorter single chain enzyme retaining the processed oligosaccharides. A pulse-chase experiment demonstrates the conversion of the precursor into the mature form. In addition, results on deglycosylation of metabolically labeled cathepsin Z are shown and the alterations in the apparent size of the glycopeptides are explained.

The Host Antimicrobial Peptide Bac71-35 Binds to Bacterial Ribosomal Proteins and Inhibits Protein Synthesis

Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340μM) inside the E.coli cytoplasm. The peptide specifically and completely inhibits invitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E.coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis.

The metabolism of aged seeds. The free and membrane-bound polyribosomes of germinating rye grains of different ages

Grains of winter rye harvested in 1976, 1978, 1982 and 1984 were studied. Free and membrane-bound polyribosomes were isolated from embryos of imbibing and germinating grains. There was no correlation between grain viability and the amout of ribosomes. The highest incorporation of radioactive precursors (both total and specific radioactivity) was found in the RNA and ribosomal proteins from the grains with the highest viability - harvested in 1984. Lower radioactivity levels were observed in the 2 to 6 year old grains. There was no incorporation of radioactive precursors into ribosomal proteins in dead seeds.

Studies for the Development of Novel Anti-MRSA/VRE Drugs

The widespread emergence of multidrug-resistant Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) is a high threat for human health. In the course of screening for active compounds against the above drug-resistant bacteria from microbial metabolites, we discovered three kinds of novel compounds designated tripropeptins, pargamicin, and amycolamicin. Tripropeptin C (TPPC), major component of tripropeptins, is the most promising compound because it is efficacious against MRSA and VRE both in vitro and in a mouse septicemia model, and shows no cross-resistance to available drugs including vancomycin. Studies of incorporation of radioactive macromolecular precursors and accumulation of UDP-MurNAc-pentapeptide in the cytoplasm in S. aureus Smith revealed that TPPC is a cell wall synthesis inhibitor. Antimicrobial activity of TPPC was weakened by addition of prenylpyrophosphates but not with prenylphosphates, UDP-linked sugars, or the pentapeptide of peptidoglycan. Direct interaction between TPPC and undecaprenyl pyrophosphate (C(55)-PP) was observed by mass spectrometry and thin layer chromatography, and TPPC inhibits C(55)-PP phosphatase, which plays a crucial role in peptidoglycan synthesis at an IC(50) of 0.03-0.1 µM in vitro. From the analysis of accumulation of lipid carrier-related compounds, TPPC caused accumulation of C(55)-PP in situ, leading to the accumulation of a glycine-added lipid intermediate, suggesting a distinct mode of action from that of clinically important drugs such as vancomycin, daptomycin, and bacitracin. TPPC might represent a promising novel class of antibiotic against MRSA and VRE infections.

Cytotoxic Effect of Dexamethasone Restricted to Noncycling, Early G1 Phase of Melanoma Cells

This study deals with the inhibitory effects of dexamethsone on the proliferation of a human melanoma cells after separation into different fractions according to their position in the cell cycle using a gravity sedimentation chamber. Fractions with high percentage of G1 cells were more susceptible to the action of dexamethsone than those with a lower percentage of G1 cells. Dexamethsone stimulated the rate of incorporation of radioactive precursors into acid-precipitable materials of melanoma M-5A cells starting 24 hours after treatment for one hour. Moreover, dexamethsone treatment markedly increased the volume of the M-5A cells with increasing the possibility of stimulating the transcriptional/translational activity in the cells. This study may hopefully stimulate the development of new approaches in systemic and/or regional chemotherapy with malignant melanoma using dexamethsone with other alkylating agents to get synergestic interaction.

Biological activities of pargamicin A, a novel cyclic peptide antibiotic from Amycolatopsis sp.

The time-kill studies using pargamicin A against Staphylococcus aureus and Enterococcus faecalis were performed. The effects of the incorporation of radioactive precursors into macromolecules, membrane potential and function using fluorescent dyes were also examined. These studies revealed that rapid bactericidal activity of pargamicin A correlates with the perturbation of bacterial cell membrane potential and membrane function.

Early responses of liver to cortisone in alloxan-diabetic rats.

When administered to alloxan-diabetic rats, cortisone elicits the same early hepatic responses which are observed in normal rats: increase of tyrosine transaminase and tryptophane oxygenase activities, increased incorporation of radioactive precursors into RNA, enhanced glycogen deposition. It is therefore likely that the Cortisol action upon liver is not dependent on the presence of insulin.

Mercury immune toxicity in harbour seals: links to in vitro toxicity.

BackgroundMercury is known to bioaccumulate and to magnify in marine mammals, which is a cause of great concern in terms of their general health. In particular, the immune system is known to be susceptible to long-term mercury exposure. The aims of the present study were (1) to determine the mercury level in the blood of free-ranging harbour seals from the North Sea and (2) to examine the link between methylmercury in vitro exposure and immune functions using seal and human mitogen-stimulated peripheral blood mononuclear cells (T-lymphocytes).MethodsTotal mercury was analysed in the blood of 22 harbour seals. Peripheral blood mononuclear cells were isolated from seals (n = 11) and from humans (n = 9). Stimulated lymphocytes of both species were exposed to functional tests (proliferation, metabolic activity, radioactive precursor incorporation) under increasing doses of methylmercury (0.1 to 10 μM). The expression of cytokines (IL-2, IL-4 and TGF-β) was investigated in seal lymphocytes by RT-PCR and by real time quantitative PCR (n = 5) at methylmercury concentrations of 0.2 and 1 μM. Finally, proteomics analysis was attempted on human lymphocytes (cytoplasmic fraction) in order to identify biochemical pathways of toxicity at concentration of 1 μM (n = 3).ResultsThe results showed that the number of seal lymphocytes, viability, metabolic activity, DNA and RNA synthesis were reduced in vitro, suggesting deleterious effects of methylmercury concentrations naturally encountered in free-ranging seals. Similar results were found for human lymphocytes. Functional tests showed that a 1 μM concentration was the critical concentration above which lymphocyte activity, proliferation and survival were compromised. The expression of IL-2 and TGF-β mRNA was weaker in exposed seal lymphocytes compared to control cells (0.2 and 1 μM). Proteomics showed some variation in the protein expression profile (e.g. vimentin).ConclusionOur results suggest that seal and human PBMCs react in a comparable way to MeHg in vitro exposure with, however, larger inter-individual variations. MeHg could be an additional cofactor in the immunosuppressive pollutant cocktail generally described in the blood of seals and this therefore raises the possibility of additional additive effects in the marine mammal immune system.

Consequences of daptomycin-mediated membrane damage in Staphylococcus aureus

The proposed lethal action of daptomycin on Staphylococcus aureus results from the loss of K(+) and membrane depolarization. However, whether these events alone cause cell death has been questioned. We sought to determine whether other consequences of daptomycin-mediated membrane damage may contribute to cell death. Previously established assays were used to evaluate the membrane damaging activity of daptomycin at a single time-point of 10 min. More detailed time-course experiments were also performed to determine the kinetics of membrane depolarization and leakage of K(+), Mg(2+) and ATP. The kinetics of inhibition of macromolecular synthesis following exposure to daptomycin were also determined by assaying the incorporation of radioactive precursors into macromolecules. Daptomycin exhibited no membrane damaging activity in single time-point assays following exposure to the antibiotic for 10 min. Kinetic analysis confirmed these results as leakage of intracellular components did not occur until 20-30 min, membrane depolarization was gradual and cells remained biosynthetically active for at least 30 min after exposure to daptomycin. Viability declined rapidly after exposure to daptomycin and appeared to precede other detectable changes. These data show that daptomycin-induced loss of Mg(2+) and ATP occurs in conjunction with the previously reported leakage of K(+) and membrane depolarization. We propose that the lethal activity of daptomycin is not simply due to loss of K(+) and probably involves more general damage to the membrane.

Antibacterial activity of the secondary metabolites from in vitro cultures of Drosera aliciae

Preparations from carnivorous plants belonging to genus Drosera (Droseraceae) were shown to possess antimicrobial properties [1]. The objective of this research was to evaluate antibacterial properties of secondary metabolites obtained from Drosera aliciae Raym.-Hamet plants grown in vitro and to examine mechanism of their antimicrobial action. Bactericidal activity of chloroform and methanol extracts from D. aliciae as well as pure ramentaceone as the major naphthoquinone present in this plant were examined against resistant and susceptible to antibiotics strains from the following species of human bacterial pathogens (hospital isolates): Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Chloroform extract proved to be more effective than methanol preparation against all of the tested strains except for P. aeruginosa isolates with the lowest minimal bactericidal concentration (MBC) values in case of S. aureus (25–50mg FW ml-1). Methanol extract from D. aliciae caused 3 and 8-fold increase (for 0.1 and 0.25 MBC respectively) in generation time of E. faecalis in relation to control, chloroform preparation elevated generation time 1.2 and 2-fold, while both tested concentrations of ramentaceone caused no statistically significant growth retardation. The influence of D. aliciae extracts and ramentaceone on the synthesis of DNA, RNA and proteins in cultures of E. faecalis was estimated by measurement of incorporation of radioactive precursors: [3H]thymidine, [3H]uridine or [3H]leucine, respectively. Methanol extract from D. aliciae except for a moderate effect on DNA synthesis had no influence on neither RNA nor protein synthesis. Chloroform preparation caused about 75% decrease in [3H]uridine incorporation into RNA in comparison to control after 60min and a significant reduction of DNA and protein synthesis (44% and 30% respectively). Ramentaceone also reduced DNA and RNA synthesis but less efficiently than chloroform extract and caused no changes in protein synthesis.

Proteomic Analysis of a Nutritional Shift-up in Saccharomyces cerevisiae Identifies Gvp36 as a BAR-containing Protein Involved in Vesicular Traffic and Nutritional Adaptation

Yeast cells undergoing a nutritional shift-up from a poor to a rich carbon source take several hours to adapt to the novel, richer carbon source. The budding index is a physiologically relevant "global" parameter that reflects the complex links between cell growth and division that are both coordinately and deeply affected by nutritional conditions. We used changes in budding index as a guide to choose appropriate, relevant time points during an ethanol to glucose nutritional shift-up for preparation of samples for the analysis of proteome by two-dimensional electrophoresis/mass spectrometry. About 600 spots were detected. 90 spots, mostly comprising proteins involved in intermediary metabolism, protein synthesis, and response to stress, showed differential expression after glucose addition. Among modulated proteins we identified a protein of previously unknown function, Gvp36, showing a transitory increase corresponding to the drop of the fraction of budded cells. A gvp36Delta strain shares several phenotypes (including general growth defects, heat shock, and high salt sensitivity, defects in polarization of the actin cytoskeleton, in endocytosis and in vacuolar biogenesis, defects in entering stationary phase upon nutrient starvation) with secretory pathway mutants and with mutants in genes encoding the two previously known yeast BAR proteins (RSV161 and RSV167). We thus propose that Gvp36 represents a novel yeast BAR protein involved in vesicular traffic and in nutritional adaptation.

Induction of Apoptosis of Human Tumor Cells by a 4,9-Diazapyrenium Derivative and Its Affects on Topoisomerase-II Action

Background: 5,10-Diphenyl-4,9-dimethyl-4,9-diazapyrenium hydrogensulfate (FDAP) is a newly synthesized DNA intercalator with strong antiproliferative activity against various human tumor cells. In this study, FDAP’s potential to induce apoptosis in human pancreatic (MIAPaCa-2) and colon (Caco-2) carcinoma cells and its effects on topoisomerase-II activity were investigated. Methods: Cytotoxicity was analyzed using the MTT assay. Antiproliferative activity was measured using a radioactive precursor incorporation assay. DNA fragmentation was analyzed by agarose gel electrophoresis. Apoptosis was detected using annexin-V fluorescein. To screen the topoisomerase-II-targeted effects of FDAP, cell-free assays were used. Results: FDAP at concentrations of 10 and 1 µM caused a strong cytotoxic effect on MIAPaCa-2 cells, but only a slight effect on WI38 cell viability. Inhibition of the DNA, RNA and protein synthesis of the treated tumor cells was dependent on cell type. The DNA of the treated MIAPaCa-2 cells was fragmented. Phosphatidylserine externalization was detected in both tumor cell lines investigated. FDAP caused generation of DNA strand breaks due to interaction with human topoisomerase-IIα. Conclusion: 1 µM FDAP caused apoptotic cell death of treated tumor cells. FDAP stimulated topoisomerase-II-mediated DNA cleavage and could be considered a topoisomerase-II poison.

Lactoferricin B inhibits bacterial macromolecular synthesis in Escherichia coli and Bacillus subtilis

Most antimicrobial peptides have an amphipathic, cationic structure, and an effect on the cytoplasmic membrane of susceptible bacteria has been postulated as the main mode of action. Other mechanisms have been reported, including inhibition of cellular functions by binding to DNA, RNA and proteins, and the inhibition of DNA and/or protein synthesis. Lactoferricin B (Lfcin B), a cationic peptide derived from bovine lactoferrin, exerts slow inhibitory and bactericidal activity and does not lyse susceptible bacteria, indicating a possible intracellular target. In the present study incorporation of radioactive precursors into DNA, RNA and proteins was used to demonstrate effects of Lfcin B on macromolecular synthesis in bacteria. In Escherichia coli UC 6782, Lfcin B induces an initial increase in protein and RNA synthesis and a decrease in DNA synthesis. After 10 min, the DNA-synthesis increases while protein and RNA-synthesis decreases significantly. In Bacillus subtilis, however, all synthesis of macromolecules is inhibited for at least 20 min. After 20 min RNA-synthesis increases. The results presented here show that Lfcin B at concentrations not sufficient to kill bacterial cells inhibits incorporation of radioactive precursors into macromolecules in both Gram-positive and Gram-negative bacteria.

Estimates of bacterial production using the leucine incorporation method are influenced by differences in protein retention of microcentrifuge tubes

The most widely used methods to determine bacterial production involve measuring the incorporation of radioactive precursors, such as leucine, into macromolecular pools. The leucine method that involves incubation and extraction within a single microcentrifuge tube has become a widely used technique because of its relative convenience, precision, and low cost. We observed a discrepancy in parallel determinations of leucine incorporation for the same water samples that lead us to explore aspects of the method including tube‐washing methods, operator differences, and differences among tube brands. Operators and washing methods had minimal effects on rate measurements, but results were strongly dependent on tube brands. Differences in tube performance were observed consistently in comparisons from a variety of freshwater and marine environments. Microcentrifuge tubes differed in protein retention with the consequence that estimates of leucine incorporation in a given sample could vary by as much as 60% depending on the tube used. There was no simple relationship between tube plastics or manufacturer and tube performance. We advise researchers to check the protein retention of tubes and to use the same brand of tube during field studies to minimize this potential source of variation.

Fibronectin metabolism of cartilage explants in response to the frequency of intermittent loading

Chondrocytes within articular cartilage experience complete unloading between loading cycles and in so doing utilize mechanical signals to regulate their own metabolic activities. A strongly elevated fibronectin content is an early feature in osteoarthritis and appears to be related to increases in both the synthesis and retention of this glycoprotein. The objectives of this study were to investigate systematically whether the frequency of intermittently applied cyclic mechanical loading of cartilage explants alters the biosynthesis and retention of fibronectin, and to assess whether it is possible to induce in vitro osteoarthritic-like changes of this metabolic parameter by mechanical means over a period of 6 days. Cartilage plugs consisting of viability-checked chondrocytes were exposed to sinusoidal cyclic compressive pressure alterations of 0.1, 0.5 or 1.0 Hz frequency with a peak stress of 0.5 MPa for a period of 5, 10 or 20 s, followed by an unloading period of 10, 100 or 1000 s, and compared to unloaded reference plugs from the same joint and topographic origin. The incorporation of radioactive precursor into fibronectin during the last 18 h, the content of fibronectin, and the viability of chondrocytes were determined. Our data revealed that (a) the fibronectin synthesis was selectively, but non-linearly affected by the frequency of intermittent loads applied (as defined by the frequency of the applied force, the duration of the loading cycle and the duration of the force-free period between each loading cycle), and that (b) the retention of endogenous fibronectin and proteins within loaded cartilage explants is strongly elevated. These data support our hypothesis that the mechanical factor “frequency of intermittent loading” seems to be the crucial mechanical parameter controlling the metabolism of chondrocytes. The effect of the frequency of intermittent loading cannot be described by a simple statistical correlation, so that no specific predictions are possible. However, our results imply that distinct loading protocols have been established that can induce alterations of the fibronectin metabolism similar to those observed in human and animal osteoarthritis.

Overexpression of the cgtA (yhbZ, obgE) gene, coding for an essential GTP-binding protein, impairs the regulation of chromosomal functions in Escherichia coli.

GTPases belonging to the Obg/Gtp1 subfamily are essential proteins in most bacterial species and are evolutionarily conservative from bacteria to humans. However, their specific functions in the regulation of cellular processes are largely unknown. Here we demonstrate that overproduction of a member of the Obg/Gtp1 subfamily, cgtA ( yhbZ, obgE) gene product, in Escherichia coli is deleterious for bacterial growth. However, syntheses of DNA, RNA, and proteins were not significantly affected under these conditions as measured by efficiency of incorporation of radioactive precursors. On the other hand, flow cytometry studies revealed that cgtA-overexpressing bacteria form enlarged cells with significantly changed distribution of chromosomal DNA. These results strongly suggest that overproduction of a GTP-binding protein from the Obg/Gtp1 subfamily impairs regulation of some chromosomal functions in E. coli, especially synchronization of DNA replication initiation and possibly also partitioning of daughter chromosomes after a replication round.