Translational regulation contributes to the elevated CO2 response in two Solanum species.

Understanding the impact of elevated CO2 (eCO2 ) in global agriculture is important given climate change projections. Breeding climate-resilient crops depends on genetic variation within naturally varying populations. The effect of genetic variation in response to eCO2 is poorly understood, especially in crop species. We describe the different ways in which Solanum lycopersicum and its wild relative S. pennellii respond to eCO2 , from cell anatomy, to the transcriptome, and metabolome. We further validate the importance of translational regulation as a potential mechanism for plants to adaptively respond to rising levels of atmospheric CO2 .

Comparison of the effects of pachymic acid, moronic acid and hydrocortisone on the polysome loading of RNAs in lipopolysaccharide-treated THP-1 macrophages.

We have proposed that analysis of ribosome-loaded mRNAs (i.e., the translatome) is useful for elucidation of pharmacological effects of phytocompounds in immune cells, regarding the involvement of post-transcriptional regulation mechanisms. In the present study, we compared the effects of pachymic acid from Poria cocos fungus and moronic acid from propolis with those of hydrocortisone on the translatomes of THP-1 macrophages exposed to bacterial lipopolysaccharide (LPS) to find clues to their biological effects. Polysome-associated RNAs collected from cells treated for 3 h with LPS plus each of the compounds were analyzed by DNA microarray followed by analyses of pathways/gene ontologies (GO). Upregulated mRNAs in enriched pathways that were found to contain AUUUA (AU)-rich motifs were checked by real-time PCR, and expression of candidate RNA-binding proteins stabilizing/destabilizing such AU-rich mRNAs was checked by Western blotting. The numbers of upregulated and downregulated genes (fold-changes ± 2.0 versus vehicle-control) were, respectively, 209 and 125 for moronic acid, 23 and 2 for pachymic acid, and 214 and 59 for hydrocortisone treatment. Overlapping with hydrocortisone treatment for upregulation were 158 genes in moronic acid and 17 in pachymic acid treatment; of these, 16 overlapped within all treatments (C-X-C motif chemokine ligands, interferon-induced protein with tetratricopeptide repeats, etc.). Pathway analyses showed GO enrichments such as 'immune response', 'receptor binding', 'extracellular space' etc. The pachymic acid-upregulated mRNAs (highly overlapped with the other 2 treatments) showed the presence of signal peptides and AU-rich motifs, suggesting regulation by AU-rich element (ARE)-binding proteins. The expression of ARE-binding protein HuR/ELAV-1 was increased by the 3 compounds, and AUF1/hnRNP D was decreased by pachymic acid. These results suggested that pachymic acid and moronic acid effects may involve as yet unknown post-transcriptional modulation via ARE-binding proteins resembling that of glucocorticoids.

SMALL AUXIN UP RNA62/75 Are Required for the Translation of Transcripts Essential for Pollen Tube Growth.

Successful pollen tube elongation is critical for double fertilization, but the biological functions of pollen tube genes and the regulatory machinery underlying this crucial process are largely unknown. A previous translatomic study revealed two Arabidopsis (Arabidopsis thaliana) SAUR (SMALL AUXIN UP RNA) genes, SAUR62 and SAUR75, whose expression is up-regulated by pollination. Here, we found that both SAUR62 and SAUR75 localized mainly to pollen tube nuclei. The siliques of homozygous saur62 (saur62/-), saur75 (saur75/-), and the SAUR62/75 RNA interference (RNAi) knockdown line had many aborted seeds. These lines had normal pollen viability but defective in vitro and in vivo pollen tube growth, with branching phenotypes. Immunoprecipitation with transgenic SAUR62/75-GFP flowers revealed ribosomal protein RPL12 family members as potential interacting partners, and their individual interactions were confirmed further by yeast two-hybrid and bimolecular fluorescence complementation assays. Polysome profiling showed reduced 80S ribosome abundance in homozygous saur62, saur75, ribosomal large subunit12c, and SAUR62/75 RNAi flowers, suggesting that SAUR62/75 play roles in ribosome assembly. To clarify their roles in translation, we analyzed total proteins from RNAi versus wild-type flowers by isobaric tags for relative and absolute quantitation, revealing significantly reduced expression of factors participating in pollen tube wall biogenesis and F-actin dynamics, which are critical for the elastic properties of tube elongation. Indeed, RNAi pollen tubes showed mislocalization of deesterified and esterified pectins and F-actin organization. Thus, the biological roles of SAUR62/75 and their RPL12 partners are critical in ribosomal pre-60S subunit assembly for efficient pollen tube elongation and subsequent fertilization.

Dynamics of the Pollen Sequestrome Defined by Subcellular Coupled Omics.

Reproduction success in angiosperm plants depends on robust pollen tube growth through the female pistil tissues to ensure successful fertilization. Accordingly, there is an apparent evolutionary trend to accumulate significant reserves during pollen maturation, including a population of stored mRNAs, that are utilized later for a massive translation of various proteins in growing pollen tubes. Here, we performed a thorough transcriptomic and proteomic analysis of stored and translated transcripts in three subcellular compartments of tobacco (Nicotiana tabacum), long-term storage EDTA/puromycin-resistant particles, translating polysomes, and free ribonuclear particles, throughout tobacco pollen development and in in vitro-growing pollen tubes. We demonstrated that the composition of the aforementioned complexes is not rigid and that numerous transcripts were redistributed among these complexes during pollen development, which may represent an important mechanism of translational regulation. Therefore, we defined the pollen sequestrome as a distinct and highly dynamic compartment for the storage of stable, translationally repressed transcripts and demonstrated its dynamics. We propose that EDTA/puromycin-resistant particle complexes represent aggregated nontranslating monosomes as the primary mediators of messenger RNA sequestration. Such organization is extremely useful in fast tip-growing pollen tubes, where rapid and orchestrated protein synthesis must take place in specific regions.

Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin.

BACKGROUND: We previously selected four strains of Saccharomyces cerevisiae for their ability to produce the aquaporin Fps1 in sufficient yield for further study. Yields from the yeast strains spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 µg/mL doxycycline) that had been transformed with an expression plasmid containing 249 base pairs of 5' untranslated region (UTR) in addition to the primary FPS1 open reading frame (ORF) were 10-80 times higher than yields from wild-type cells expressing the same plasmid. One of the strains increased recombinant yields of the G protein-coupled receptor adenosine receptor 2a (A2aR) and soluble green fluorescent protein (GFP). The specific molecular mechanisms underpinning a high-yielding Fps1 phenotype remained incompletely described. RESULTS: Polysome profiling experiments were used to analyze the translational state of spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 µg/mL doxycycline); all but gcn5Δ were found to exhibit a clear block in translation initiation. Four additional strains with known initiation blocks (rpl31aΔ, rpl22aΔ, ssf1Δ and nop1Δ) also improved the yield of recombinant Fps1 compared to wild-type. Expression of the eukaryotic transcriptional activator GCN4 was increased in spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 µg/mL doxycycline); these four strains also exhibited constitutive phosphorylation of the eukaryotic initiation factor, eIF2α. Both responses are indicative of a constitutively-stressed phenotype. Investigation of the 5'UTR of FPS1 in the expression construct revealed two untranslated ORFs (uORF1 and uORF2) upstream of the primary ORF. Deletion of either uORF1 or uORF1 and uORF2 further improved recombinant yields in our four strains; the highest yields of the uORF deletions were obtained from wild-type cells. Frame-shifting the stop codon of the native uORF (uORF2) so that it extended into the FPS1 ORF did not substantially alter Fps1 yields in spt3Δ or wild-type cells, suggesting that high-yielding strains are able to bypass 5'uORFs in the FPS1 gene via leaky scanning, which is a known stress-response mechanism. Yields of recombinant A2aR, GFP and horseradish peroxidase could be improved in one or more of the yeast strains suggesting that a stressed phenotype may also be important in high-yielding cell factories. CONCLUSIONS: Regulation of Fps1 levels in yeast by translational control may be functionally important; the presence of a native uORF (uORF2) may be required to maintain low levels of Fps1 under normal conditions, but higher levels as part of a stress response. Constitutively-stressed yeast strains may be useful high-yielding microbial cell factories for recombinant protein production.

Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function.

Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki = 3.74 ± 1.63 nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells.

Yeast m6A Methylated mRNAs Are Enriched on Translating Ribosomes during Meiosis, and under Rapamycin Treatment.

Interest in mRNA methylation has exploded in recent years. The sudden interest in a 40 year old discovery was due in part to the finding of FTO's (Fat Mass Obesity) N6-methyl-adenosine (m6A) deaminase activity, thus suggesting a link between obesity-associated diseases and the presence of m6A in mRNA. Another catalyst of the sudden rise in mRNA methylation research was the release of mRNA methylomes for human, mouse and Saccharomyces cerevisiae. However, the molecular function, or functions of this mRNA 'epimark' remain to be discovered. There is supportive evidence that m6A could be a mark for mRNA degradation due to its binding to YTH domain proteins, and consequently being chaperoned to P bodies. Nonetheless, only a subpopulation of the methylome was found binding to YTHDF2 in HeLa cells.The model organism Saccharomyces cerevisiae, has only one YTH domain protein (Pho92, Mrb1), which targets PHO4 transcripts for degradation under phosphate starvation. However, mRNA methylation is only found under meiosis inducing conditions, and PHO4 transcripts are apparently non-methylated. In this paper we set out to investigate if m6A could function alternatively to being a degradation mark in S. cerevisiae; we also sought to test whether it can be induced under non-standard sporulation conditions. We find a positive association between the presence of m6A and message translatability. We also find m6A induction following prolonged rapamycin treatment.

Translation attenuation via 3' terminal codon usage in bovine csn1s2 is responsible for the difference in αs2- and ß-casein profile in milk.

The rate of secretion of αs2-casein into bovine milk is approximately 25% of that of ß-casein, yet mammary expression of their respective mRNA transcripts (csn1s2 and csn2) is not different. Our objective was to identify molecular mechanisms that explain the difference in translation efficiency between csn1s2 and csn2. Cell-free translational efficiency of csn2 was 5 times that of csn1s2. Transcripts of csn1s2 distributed into heavier polysomes than csn2 transcripts, indicating an attenuation of elongation and/or termination. Stimulatory and inhibitory effects of the 5' and 3' UTRs on translational efficiency were different with luciferase and casein sequences in the coding regions. Substituting the 5' and 3' UTRs from csn2 into csn1s2 did not improve csn1s2 translation, implicating the coding region itself in the translation difference. Deletion of a 28-codon fragment from the 3' terminus of the csn1s2 coding region, which displays codons with low correlations to cell fitness, increased translation to a par with csn2. We conclude that the usage of the last 28 codons of csn1s2 is the main regulatory element that attenuates its expression and is responsible for the differential translational expression of csn1s2 and csn2.

[Effect of structural P-protein of potato curly dwarf virus on replicase activity of mRNP-complexes of infected plants].

All representatives of rhabdoviruses contain a nucleocapside phosphoprotein - P-protein which is an essential subunit of the viral RNA-dependent RNA polymerase complex. As a result of studying the effect of nucleocapside protein P(NS) on replicase activity of mRNP isolated from plants infected by potato curly dwarf virus in the system in vitro, it was established that nucleocapside P-protein stimulates considerably the replicase activity of membrane-bound polysomal m-RNP P-protein being available in concentration of 15 microg/ml in the replication system in vitro of membrane-bound polysomal mRNP, the replicase activity increased 11.7 times. This property of nucleocapside P-protein at the same concentration was displayed to a less extent with the presence of free polysomal mRNP, in the system in vitro. Thus the replicase activity mRNP-complexes in the replication system in vitro depends on the presence of nucleocapside viral P-protein in the system. Its concentration being increased or decreased, one can observe the change of the replicase activity.

The two Arabidopsis RPS6 genes, encoding for cytoplasmic ribosomal proteins S6, are functionally equivalent.

Many eukaryotic genomes have experienced ancient whole-genome duplication (WGD) followed by massive gene loss. These eliminations were not random since some gene families were preferentially retained as duplicates. The gene balance hypothesis suggests that those genes with dosage reduction can imbalance their interacting partners or complex, resulting in decreased fitness. In Arabidopsis, the cytoplasmic ribosomal proteins (RP) are encoded by gene families with at least two members. We have focused our study on the two RPS6 genes in an attempt to understand why they have been retained as duplicates. We demonstrate that RPS6 function is vital for the plant. We also show that reducing the level of RPS6 accumulation (in the knock-out rps6a or rps6b single mutants, or in the double heterozygous RPS6A/rps6a,RPS6B/rps6b), confers a slow growth phenotype (haplodeficiency). Importantly, we demonstrate that the functions of two RPS6 genes are redundant and interchangeable. Finally, like in most other described Arabidopsis rp mutants, we observed that a reduced RPS6 level slightly alters the dorsoventral leaf patterning. Our results support the idea that the Arabidopsis RPS6 gene duplicates were evolutionarily retained in order to maintain an expression level necessary to sustain the translational demand of the cell, in agreement with the gene balance hypothesis.

Ultrastructural changes in tumor cells treated with liposomal forms of anticancer drugs.

AIM: To determine the main ultrastructural changes in MCF-7 sublines sensitive and resistant to cytotoxic action of anticancer drugs, resulting from the treatment with conventional and liposomal forms of cisplatin and doxorubicin. METHODS: Electron microscopy, light microscopy, MTT-test. RESULTS: It has been shown that the phenomenon of drug resistance is associated with complication of ultrastructural organization of cells and more high differentiation by the main cytomorphologic characteristics which promote their resistance to cytotoxic action of anticancer preparations. Cytoarchitectonics of all resistant cells possesses common patterns and doesn't depend on the particular drugs toward which the resistance has been developed. It has been shown that the cells of the parental form MCF-7 line are more sensitive to cytotoxic action of doxorubicin than to cisplatin. Liposomal forms of anticancer drugs used at the same concentrations that the conventional ones, especially that of doxorubicin, caused more expressed alterations in ultrastructural organization of cells of all studied sublines with dominance of apoptotic processes. CONCLUSION: Evaluating an effect of equal concentrations of cisplatin and doxorubicin in conventional and liposomal forms, one may conclude on higher cytotoxic action of doxorubicin vs. cisplatin that is expressed in a wider spectrum of ultrastructural changes of cell architectonics in different sublines of MCF-7 cells and higher rate of apoptosis.

Identification of actively translated mRNA transcripts in a rat model of early-stage colon carcinogenesis.

With respect to functional mapping of gene expression signatures, the steady-state mRNA expression level does not always accurately reflect the status of critical signaling proteins. In these cases, control is exerted at the epigenetic level of recruitment of mRNAs to polysomes, the factories of ribosomes that mediate efficient translation of many cellular messages. However, to date, a genome-wide perspective of the effect of carcinogen and chemoprotective bioactive diets on actively translated (polysomal) mRNA populations has not been done. Therefore, we used an established colon cancer model, i.e., the azoxymethane (AOM)-treated rat, in combination with a chemoprotective diet extensively studied in our laboratory, i.e., n-3 polyunsaturated fatty acids, to characterize the molecular processes underlying the transformation of normal colonic epithelium. The number of genes affected by AOM treatment 10 weeks after carcinogen injection was significantly greater in the polysome RNA fraction compared with the total RNA fraction as determined using a high-density microarray platform. In particular, polysomal loading patterns of mRNAs associated with the Wnt-beta catenin, phospholipase A(2)-eicosanoid and the mitogen-activated protein kinase signaling axes were significantly upregulated at a very early period of tumor development in the colon. These data indicate that translational alterations are far more extensive relative to transcriptional alterations in mediating malignant transformation. In contrast, transcriptional alterations were found to be more extensive relative to translational alterations in mediating the effects of diet. Therefore, during early stage colonic neoplasia, diet and carcinogen seem to predominantly regulate gene expression at multiple levels via unique mechanisms.

Genome-wide analysis of plastid gene expression in potato leaf chloroplasts and tuber amyloplasts: transcriptional and posttranscriptional control.

Gene expression in nongreen plastids is largely uncharacterized. To compare gene expression in potato (Solanum tuberosum) tuber amyloplasts and leaf chloroplasts, amounts of transcripts of all plastid genes were determined by hybridization to plastome arrays. Except for a few genes, transcript accumulation was much lower in tubers compared with leaves. Transcripts of photosynthesis-related genes showed a greater reduction in tubers compared with leaves than transcripts of genes for the genetic system. Plastid genome copy number in tubers was 2- to 3-fold lower than in leaves and thus cannot account for the observed reduction of transcript accumulation in amyloplasts. Both the plastid-encoded and the nucleus-encoded RNA polymerases were active in potato amyloplasts. Transcription initiation sites were identical in chloroplasts and amyloplasts, although some differences in promoter utilization between the two organelles were evident. For some intron-containing genes, RNA splicing was less efficient in tubers than in leaves. Furthermore, tissue-specific differences in editing of ndh transcripts were detected. Hybridization of the plastome arrays with RNA extracted from polysomes indicated that, in tubers, ribosome association of transcripts was generally low. Nevertheless, some mRNAs, such as the transcript of the fatty acid biosynthesis gene accD, displayed relatively high ribosome association. Selected nuclear genes involved in plastid gene expression were generally significantly less expressed in tubers than in leaves. Hence, compared with leaf chloroplasts, gene expression in tuber amyloplasts is much lower, with control occurring at the transcriptional, posttranscriptional, and translational levels. Candidate regulatory sequences that potentially can improve plastid (trans)gene expression in amyloplasts have been identified.

Genome-wide analysis of mRNA polysomal profiles with spotted DNA microarrays.

The sedimentation of an mRNA in sucrose gradients is highly affected by its ribosomal association. Sedimentation analysis has therefore become routine for studying changes in ribosomal association of mRNAs of interest. DNA microarray technology has been combined with sedimentation analysis to characterize changes in ribosomal association for thousands of mRNAs in parallel. Such analyses revealed mRNAs that are translationally regulated and have provided new insights into the translation process. In this chapter, we describe possible experimental designs for analyzing genome-wide changes in ribosomal association, and discuss some of their advantages and disadvantages. We then provide a detailed protocol for analysis of polysomal fractions using spotted DNA microarrays.

Translation of non-capped mRNAs in a eukaryotic cell-free system: acceleration of initiation rate in the course of polysome formation.

Real-time monitoring of the translation of non-capped luciferase mRNA in a wheat germ cell-free system has been performed by continuous in situ measurement of the luminescence increase in the translation mixture. The phenomenon of acceleration of translation has been revealed. It has been shown that the acceleration is accompanied by the loading of translating polysomes with additional ribosomes, and thus is caused mainly by a rise in the initiation rate, rather than the stimulation of elongation or the involvement of additional mRNA molecules in translation. The acceleration requires a sufficient concentration of mRNA and depends on the sequence of the 5' untranslated region (UTR). It can be abolished by the addition of excess cap analog (m(7)GpppGm). As the acceleration does not depend on the preliminary translation of other mRNAs in the same extract, the conclusion has been made that the effect is not due to activation of the ribosome population or other components of the system during translation, but rather it is the consequence of intra-polysomal events. The acceleration observed is discussed in terms of the model of two overlapping initiation pathways in eukaryotic polysomes: translation of non-capped mRNAs starts with eIF4F-independent initiation at 5' UTR, and after the formation of sufficiently loaded polysomes, they rearrange in such a way that a mechanism of re-initiation of terminating ribosomes switches on. The eIF4F-mediated circularization of polysomes may be considered as a possible event that leads to the re-initiation switch and the resultant acceleration effect.

Analysis of gene expression in amyloplasts of potato tubers.

Gene expression in amyloplasts derived from potato tubers was analyzed at the levels of transcription, mRNA accumulation and formation of polysomes. Compared with chloroplasts, overall transcriptional activity is considerably reduced in amyloplasts. Nevertheless, several transcripts are synthesized in amyloplasts during growth of tubers. Among the transcribed amyloplast genes are the ribosomal operon and the psbA gene. Primer extension analysis provided evidence that in amyloplasts the plastid encoded RNA polymerase (PEP) is the principal RNA polymerase involved in the transcription of the rrn operon. Analysis of plastid steady state transcripts showed that there are only small differences in the levels of specific transcripts between amyloplasts and chloroplasts. With respect to the low transcription rate of the accumulating RNA-species in amyloplasts, a high stability of these transcripts is obvious. Though amyloplasts possess polysomes, specific mRNAs associated with such polysomes could not be detected. This suggests that translation could be impaired in amyloplasts, which, in turn, implies that these organelles are not suitable targets for the expression of transgenes introduced into the plastid genome by plastid transformation.

Selenium treatment protects diabetes-induced biochemical and ultrastructural alterations in liver tissue.

We have shown that a single dose of streptozotocin (STZ) (50 mg/kg body weight) injected into rats caused significant changes in some antioxidant enzyme activities, such as glutathione peroxidase, glutathione reductase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase activities, and acid-soluble sulfhydryl levels of the liver tissue with respect to the control rats. Furthermore, these alterations in the activities of the antioxidant enzymes were accompanied by significant changes in the ultrastructure of the liver tissue; mainly intercellular biliary canaliculi were distended and contained stagnant bile, swollen mitochondria in hepatocytes and disoriented and disintegrating cristae, dilatation of the rough endoplasmic reticulum (rER) with detachment of ribosomes, and dissociation of polysomes. Both diabetic and normal rats were treated with sodium selenite (5 micromol/kg/d, intra peritoneally) for 4 wk following 1 wk of diabetes induction. This treatment of diabetic rats improved significantly diabetes-induced alterations in liver antioxidant enzymes. Moreover, treating of diabetic rats with sodium selenite prevented primarily the variation in staining quality of hepatocytes nuclei, increased density and eosinophilia of the cytoplasm, focal sinusoidal dilatation and congestion, and increased numbers of mitochondria with different size and shape. In summary, treatment of diabetic rats with sodium selenite has beneficial effects on both antioxidant system and the ultrastructure of the liver tissue. These findings suggest that diabetes-induced oxidative stress can be responsible for the development of diabetic complications and antioxidant treatment can protect the target organs against diabetes.

Activated polymorphonuclear leukocytes rapidly synthesize retinoic acid receptor-alpha: a mechanism for translational control of transcriptional events.

In addition to releasing preformed granular proteins, polymorphonuclear leukocytes (PMNs) synthesize chemokines and other factors under transcriptional control. Here we demonstrate that PMNs express an inducible transcriptional modulator by signal-dependent activation of specialized mechanisms that regulate messenger RNA (mRNA) translation. HL-60 myelocytic cells differentiated to surrogate PMNs respond to activation by platelet activating factor by initiating translation and with appearance of specific mRNA transcripts in polyribosomes. cDNA array analysis of the polyribosome fraction demonstrated that retinoic acid receptor (RAR)-alpha, a transcription factor that controls the expression of multiple genes, is one of the polyribosome-associated transcripts. Quiescent surrogate HL60 PMNs and primary human PMNs contain constitutive message for RAR-alpha but little or no protein. RAR-alpha protein is rapidly synthesized in response to platelet activating factor under the control of a specialized translational regulator, mammalian target of rapamycin, and is blocked by the therapeutic macrolide rapamycin, events consistent with features of the 5' untranslated region of the transcript. Newly synthesized RAR-alpha modulates production of interleukin-8. Rapid expression of a transcription factor under translational control is a previously unrecognized mechanism in human PMNs that indicates unexpected diversity in gene regulation in this critical innate immune effector cell.

Eukaryotic protein synthesis inhibitors identified by comparison of cytotoxicity profiles.

The National Cancer Institute (NCI) Human Tumor Cell Line Anti-Cancer Drug Screen has evaluated the cytotoxicity profiles of a large number of synthetic compounds, natural products, and plant extracts on 60 different cell lines. The data for each compound/extract can be assessed for similarity of cytotoxicity pattern, relative to a given test compound, using an algorithm called COMPARE. In applying a chemical biology approach to better understand the mechanism of eukaryotic protein synthesis, we used these resources to search for novel inhibitors of translation. The cytotoxicity profiles of 31 known protein synthesis inhibitors were used to identify compounds from the NCI database with similar activity profiles. Using this approach, two natural products, phyllanthoside and nagilactone C, were identified and characterized as novel protein synthesis inhibitors. Both compounds are specific for the eukaryotic translation apparatus, function in vivo and in vitro, and interfere with translation elongation. Our results demonstrate the feasibility of utilizing cytotoxicity profiles to identify new inhibitors of translation.

Growth Inhibitory Effects of Various Herbal Extracts on Metronidazole Resistant Strain of Trichomonas vaginalis / 감염과화학요법

BACKGROUND: Metronidazole has been known as the most effective drug for treatment of Trichomonas vaginalis-related diseases. However, it has been reported that metronidazole has adverse effects and incidence of metronidazole-resistant T. vaginalis (CDC085) has increased. Development of new drug, which is effective against metronidazole-resistant T. vaginalis and showing no adverse effects, has been required. METHODS: The purpose of this study was to investigate effects of various extracts from herbs such as Quisqualis indica, Gleditsia sinensis, Prunus armeniaca, Morus alba, Platycodon grandiflorum, Ailanthus altissima, Stemona japonica, Biota orientalis, Dryobalanops aromatica, and Cimicifuga heracleifolia on metronidazole resistant strain of T. vaginalis in vitro (CDC085). RESULTS: Anti-Trichomonas activities were observed in T. vaginalis treated with G. sinensis, P. armeniaca, and P. grandiflorum on the growth and fine structure of metronidazole resistant strain of T. vaginalis. Of the three standard extracts that showed the most effective anti-trichomonas activity, G. sinensis was the most effective. The inhibitory effects of fraction extracts of this drug were shown on the growth of T. vaginalis. The fine structure of the cytoplasm was changed after application of G. sinensis extract. The number of polyribosome and hydrogenosome decreased whereas the number of food vacuole and vacuole in the cytoplasm increased, compared with that of untreated control group. CONCLUSION: The results of our study indicate that G. sinensis may induce the inhibition of cell multiplication as well as impairment of protein synthesis of metronidazole resistant strain of T. vaginalis in vitro.