Stops Protein Synthesis Research Articles

Potential Piperolactam A Isolated From Piper betle as Natural Inhibitors of Brucella Species Aminoacyl-tRNA Synthetase for Livestock Infections: In Silico Approach.

Brucellosis is an important global zoonosis caused by the bacterium Brucella sp. Brucellosis causes abortions, reproductive failure and reduced milk production, resulting in significant economic losses. Brucella species are reported to be resistant to antibiotics, which makes treatment difficult. The urgency of discovering new drug candidates to combat Brucella's infection necessitates the exploration of novel alternative agents with unique protein targets. Aminoacyl-tRNA synthetases (aaRSs), which have fundamental functions in translation, inhibit this process, stop protein synthesis and ultimately inhibit bacterial growth. The purpose of this study was to isolate piperolactam A compounds from the methanol extract of Piper betle leaves that have potential as antibacterials to inhibit the growth of Brucella sp. causing brucellosis in livestock and to analyse the mechanism of inhibitory activity of piperolactam A compounds against the aaRS enzyme through a molecular docking approach in silico. Piperolactam A was isolated from P. betle by column chromatography and characterized by UV, IR, 1D and 2D NMRs and MS, then tested for their inhibition mechanism against the enzymes threonyl-tRNA synthetase, leucyl-tRNA synthetase (LeuRS) and methionyl-tRNA synthetase in silico. The result in silico test is that piperolactam A has the potential to inhibit LeuRS enzyme with the greater binding affinity.

Gene expression profiles for apoptotic and necrotic pathways during Amanita phalloides intoxication in mice

Background and Aims: Amanita phalloides is the deadliest toxic mushroom in the world and causes death from acute liver failure. α-amanitin (α-AMA), the most potent toxin, inhibits RNA polymerase II in hepatocytes, stops protein synthesis, and causes hepatotoxicity. However, the information about the mechanisms underlying hepatotoxicity caused by α-AMA is quite inadequate. This study aims to reveal the complex necrotic and apoptotic mechanisms occurring in mouse hepatocytes depending on A. phalloides exposure time in vivo. Methods: BALB-c male mice were divided into 5 groups (n=7): control, α-AMA-2, α-AMA-12, α-AMA-72, and α-AMA-96 groups. A poisoning model was created by oral administration of A. phalloides mushroom extract containing 10 mg/kg of α-AMA to mice and they were sacrificed after 2, 12, 72, and 96 h. Then, TNF-α, Bax, caspase-3, and Bcl-2 gene expression levels in liver tissues were examined by the RT-qPCR method. Time-dependent damage to liver tissues was also evaluated histopathologically. Results: RT-qPCR results showed that proinflammatory cytokine TNF-α mRNA expression levels increased in mouse liver tissues at 2 and 12 h after A. phalloides administration compared among the groups. Bax mRNA expression levels increased in the 12 and 72 h after A. phalloides ingestion. It was observed that caspase-3 mRNA expression levels increased in the 72 and 96 h groups compared among the groups, while Bcl-2 mRNA expression levels decreased in the 72 and 96 h groups. Conclusion: Our findings showed that necrotic mechanisms develop in the early period after A. phalloides mushroom poisoning, and then apoptotic mechanisms are effective. In conclusion, understanding the mechanisms of A. phalloides-induced hepatotoxicity will provide important information for new treatment strategies to be developed.

Differential Behavior of 4E‐BP1 and 4E‐BP2 Under ER Stress

eIF4E‐binding proteins (4E‐BPs) are a family of translational repressors, when unphosphorylated or hypo‐phosphorylated, can bind to eIF4E and inhibit eukaryotic cap‐dependent translation initiation. The 3 4E‐BPs (4E‐BP1, 4E‐BP2 and 4E‐BP3) are not isoforms but are often regarded alike in terms of inhibiting translation initiation. They are not clearly differentiated in terms of function. Not only do they originate from different genes on different chromosomes, but they also have distinct mRNA structure. We hypothesized a potential for their differential regulation under cell stress. During cell stress, 4E‐BPs need to be upregulated to stop protein synthesis and conserve cellular energy. 4E‐BP1 promoter has CARE (C/Ebp‐ATF Response Element) in its promoter region, that helps it respond to ER stress in an ATF4‐dependent manner. 4E‐BP2 on the other hand, does not have any CARE elements in its promoter, hence ideally should remain unchanged under ER stress. Gene expression was observed through qPCR and protein levels were studied using western blotting. We observed a nearly 4‐fold increase in 4E‐BP1 mRNA levels under ER stress in sync with ATF4 mRNA levels, whereas 4E‐BP2 mRNA levels didn’t change significantly. 4E‐BP1 protein levels increased under ER stress whereas 4E‐BP2 protein levels decreased under ER stress. We observed selective proteasomal degradation of 4E‐BP2 under ER stress, which recovered when treated with proteasome inhibitor MG132. The reason behind this selective degradation needs to be further elucidated. These findings shed a new light on the behavior of 4E‐BP2 under cell stress which has been, until now, treated synonymous to 4E‐BP1 in the cellular landscape.

Helicobacter pylori infection impairs chaperone-assisted maturation of Na-K-ATPase in gastric epithelium.

Helicobacter pylori infection always induces gastritis, which may progress to ulcer disease or cancer. The mechanisms underlying mucosal injury by the bacteria are incompletely understood. Here, we identify a novel pathway for H. pylori-induced gastric injury, the impairment of maturation of the essential transport enzyme and cell adhesion molecule, Na-K-ATPase. Na-K-ATPase comprises α- and β-subunits that assemble in the endoplasmic reticulum (ER) before trafficking to the plasma membrane. Attachment of H. pylori to gastric epithelial cells increased Na-K-ATPase ubiquitylation, decreased its surface and total levels, and impaired ion balance. H. pylori did not alter degradation of plasmalemma-resident Na-K-ATPase subunits or their mRNA levels. Infection decreased association of α- and β-subunits with ER chaperone BiP and impaired assembly of α/β-heterodimers, as was revealed by quantitative mass spectrometry and immunoblotting of immunoprecipitated complexes. The total level of BiP was not altered, and the decrease in interaction with BiP was not observed for other BiP client proteins. The H. pylori-induced decrease in Na-K-ATPase was prevented by BiP overexpression, stopping protein synthesis, or inhibiting proteasomal, but not lysosomal, protein degradation. The results indicate that H. pylori impairs chaperone-assisted maturation of newly made Na-K-ATPase subunits in the ER independently of a generalized ER stress and induces their ubiquitylation and proteasomal degradation. The decrease in Na-K-ATPase levels is also seen in vivo in the stomachs of gerbils and chronically infected children. Further understanding of H. pylori-induced Na-K-ATPase degradation will provide insights for protection against advanced disease.NEW & NOTEWORTHY This work provides evidence that Helicobacter pylori decreases levels of Na-K-ATPase, a vital transport enzyme, in gastric epithelia, both in acutely infected cultured cells and in chronically infected patients and animals. The bacteria interfere with BiP-assisted folding of newly-made Na-K-ATPase subunits in the endoplasmic reticulum, accelerating their ubiquitylation and proteasomal degradation and decreasing efficiency of the assembly of native enzyme. Decreased Na-K-ATPase expression contributes to H. pylori-induced gastric injury.

Thallium(I) treatment induces nucleolar stress to stop protein synthesis and cell growth

Thallium is considered as an emergent contaminant owing to its potential use in the superconductor alloys. The monovalent thallium, Tl(I), is highly toxic to the animals as it can affect numerous metabolic processes. Here we observed that Tl(I) decreased protein synthesis and phosphorylated eukaryotic initiation factor 2α. Although Tl(I) has been shown to interact with the sulfhydryl groups of proteins and cause the accumulation of reactive oxygen species, it did not activate endoplasmic reticulum stress. Notably, the level of 60S ribosomal subunit showed significant under-accumulation after the Tl(I) treatment. Given that Tl(I) shares similarities with potassium in terms of the ionic charge and atomic radius, we proposed that Tl(I) occupies certain K+-binding sites and inactivates the ribosomal function. However, we observed neither activation of ribophagy nor acceleration of the proteasomal degradation of 60S subunits. On the contrary, the ribosome synthesis pathway was severely blocked, i.e., the impairment of rRNA processing, deformed nucleoli, and accumulation of 60S subunits in the nucleus were observed. Although p53 remained inactivated, the decreased c-Myc and increased p21 levels indicated the activation of nucleolar stress. Therefore, we proposed that Tl(I) interfered the ribosome synthesis, thus resulting in cell growth inhibition and lethality.

2348 Lafora disease premature termination codons (PTCs) are likely candidates for suppression by aminoglycosides

OBJECTIVES/SPECIFIC AIMS: A small molecule therapy is within reach to treat a molecular mechanism known to result in thousands of fatal diseases. For 10% of patients with a genetic disease, a nonsense/STOP mutation/premature termination codon (PTC) is the underlying cause of their malady. PTCs prematurely stop protein synthesis and yield truncated proteins. Truncated proteins typically provide little to no proper function or activity and are rapidly degraded; thus, disease is imminent. Recent work has demonstrated that small molecules including aminoglycosides can cause the ribosome to readthrough these PTCs. Thus, PTC readthrough with small molecules is a very attractive approach for treating diseases caused by PTCs. Small molecules that promote readthrough act on the ribosome and induce a ribosomal conformational change. In this conformation, the PTC is not recognized by the translational machinery and an amino acid is incorporated into the growing peptide chain, thus protein synthesis continues and does not stop. The use of a single small molecule to readthrough various PTC mutations has been repeatedly effective for in vitro studies and some of these have progressed to clinical trials. Although there has been success in defining these small molecules, the field has discovered that every PTC is unique and likely requires a different small molecule. Thus, developing a cell culture model to test read-through of Lafora PTCs and the functionality of the protein product is the first step to developing a readthrough therapy for a LD. METHODS/STUDY POPULATION: Method for in vitro quantification of readthrough: 24 hours before transfection, HEK293 cells were split in 6-well plates. On the following day, approximately 60% confluence, the cells were transiently transfected with the WT or PTC mutated constructs using Polyethylenimine HCl MAX. Cells were transfected with a total amount of 0.35 μg DNA/well and 2 μl Polyethylenimine HCl MAX/well. Four hours later, the transfection medium was removed and replaced with fresh medium, without streptomycin and penicillin. The fresh media contained gentamicin diluted to the indicated concentration per well. Fresh gentamicin-containing medium was replaced after 24 hours. After 48 hours, lysates were collected in 100 μL mRIPA supplemented with protease inhibitors for each construct. The lysates were run on a western blot and the N-terminal was probed with anti-FLAG. A malachite green phosphatase assay to measure inorganic phosphate release from phospho-glucans, that is glycogen or LBs. Glycogen is used in this laforin bioassay as the biologically relevant substrate in order to determine the specific activity of the readthrough products. All reactions are incubated for 40 minute the absorbance is measured at 620 nm and the pmoles of phosphate released/min/nmol protein was calculated using a standard curve. RESULTS/ANTICIPATED RESULTS: HEK293 cells were transfected with MeCP2 R241X, laforin R241X, or laforin WT NT-FLAG construct, treated with different concentrations of gentamicin for 48 hours, and laforin levels were assessed by Western analysis with anti-FLAG. HEK293 cells were transfected with WT laforin or a laforin PTC CT-FLAG construct, treated with different concentrations of gentamicin for 48 hours, and laforin levels were assessed by Western analysis with anti-FLAG. B. Quantification of read-through for PTC experiments. *p-value≤0.001. #p-value≤0.001. Schematic of laforin bioassay. The assay has been performed with human and mouse tissue as well as cultured cells. B. Laforin bioassay results using laforin from PTC experiment. **p-value≤0.001. *p-value≤0.01. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that gentamicin is not only responsible for inducing readthrough of the PTC mutations, but also for promoting translation of fully functional laforin. Therefore, our in vitro system for the analysis of PTC readthrough of laforin will be useful for determining which PTC mutations are suppressible with gentamicin or other small molecules, in what quantities laforin is recovered from PTC mutations, and if the protein products possess the appropriate enzymatic function.

Mathematical analysis of codons that stop protein synthesis

A mathematical analysis was performed, which shows that the maximum length of DNA in which translation is possible for at least one of three reading frames for any DNA strand is 10 nucleotides. In the case of any nonstandard stop codon triplet, this number can only decrease. Moreover, for any DNA consisting of at most 11 nucleotides, two and more genes overlaps in plus and minus strands are possible. The probability that a stop codon triplet chosen at random belongs to the group of stop codon triplets with these properties is less than 0.06.

Overproduction of Erythromycin by Ultraviolet Mutagenesis and Expression of ermE Gene in Saccharopolyspora erythraea.

Erythromycin is a macrolide antibiotic with broad-spectrum activity against gram-positive bacteria that stops protein synthesis by binding to 50s ribosomal subunit. Classical and recombinant strain improvement, such as application of ultraviolet (UV) mutagenesis and selection of overproduction mutant, is the most important and convenient method in enhancement of antibiotic production. In the present study, Saccharopolyspora erythraea was mutagenized using UV lights and selection by tylosin resistance mutant to improve yield of erythromycin. In other sides, to improve the erythromycin yield in mutant, effects of various parameters such as carbon concentration and ermE gene expression were analyzed. In primary selection, high erythromycin producing strains and high erythromycin producer mutant were isolated by plaque agar, and an increase of 87% was observed in tylosin resistance mutant compared to wild-type strain. In secondary selection, a mutant strain (RHU233) with a production of 1.39 mg erythromycin per mL was isolated in fermentation process, which was 20 times more productive than the wild type. In contrast, it was found that glycerol can be used as an alternate carbon source in enhancement of erythromycin production. Comparison of ermE gene expression in mutants RHU233 high producer mutant RHU233 and wild type in Escherichia coli detected in accumulation of soluble hexahistidine-ermE was up to 45% of total cell protein after 18 h in mutants RHU233. Metal-chelation chromatography yielded 126 mg of hexahistidine-ermE per liter of culture with a purity slightly >95% in mutants RHU233. Finally, these optimized conditions could be used for the commercial production of this unique antibiotic.

Rapid RNase L–driven arrest of protein synthesis in the dsRNA response without degradation of translation machinery

Mammalian cells respond to double-stranded RNA (dsRNA) by activating a translation-inhibiting endoribonuclease, RNase L. Consensus in the field indicates that RNase L arrests protein synthesis by degrading ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs). However, here we provide evidence for a different and far more efficient mechanism. By sequencing abundant RNA fragments generated by RNase L in human cells, we identify site-specific cleavage of two groups of noncoding RNAs: Y-RNAs, whose function is poorly understood, and cytosolic tRNAs, which are essential for translation. Quantitative analysis of human RNA cleavage versus nascent protein synthesis in lung carcinoma cells shows that RNase L stops global translation when tRNAs, as well as rRNAs and mRNAs, are still intact. Therefore, RNase L does not have to degrade the translation machinery to stop protein synthesis. Our data point to a rapid mechanism that transforms a subtle RNA cleavage into a cell-wide translation arrest.

Analysis of Mannose 6-Phosphate Uncovering Enzyme Mutations Associated with Persistent Stuttering

GlcNAc-1-phosphodiester-N-acetylglucosaminidase ("uncovering enzyme" (UCE); EC 3.1.4.45) is a Golgi enzyme that mediates the second step in the synthesis of the mannose 6-phosphate lysosomal targeting signal on acid hydrolases. Recently, three mutations (two missense and one deletion/frameshift) in the NAGPA gene that encodes UCE have been identified in individuals with persistent stuttering. We now demonstrate that each mutation leads to lower cellular UCE activity. The p.R328C mutation impairs folding in the endoplasmic reticulum, resulting in degradation of a significant portion by the proteasomal system. The p.H84Q mutation also impairs folding and, in addition, decreases the specific activity of the enzyme that folds sufficiently to traffic to the Golgi. The p.F513SfsX113 frameshift mutation adds 113 amino acids to the C terminus of the cytoplasmic tail of the protein, including a VWLL sequence that causes rapid degradation via the proteasomal system. These biochemical findings extend the genetic data implicating mutations in the NAGPA gene in the persistent stuttering phenotype.

The AMPD1 C34T mutation is not associated with the status of Israeli athletes

The adenosine monophosphate deaminase (AMPD1) gene seems to be an important regulator of skeletal muscle energy metabolism during exercise. A nonsense C34T mutation in the AMPD1 gene results in a premature stop codon, thus stopping protein synthesis. The aim of this study was to determine the AMPD1 C34T mutation frequency distribution among 155 Israeli athletes (endurance athletes, n=74; sprinters, n=81) and 142 healthy controls. Genotyping of the AMPD1 C34T (rs5810761) mutation was performed using polymerase chain reaction (PCR). Results showed that the genotype subtype did not differ by gender in the athletes (P=0.18) or the controls (P=0.79). The AMPD1 C34T genotype distribution was in line with Hardy-Weinberg equilibrium within all groups (P>0.05). The genotype distribution and allele frequencies were similar in the groups of endurance athletes, sprinters, and controls (P=0.455). Similarly, no significant differences were observed between the subgroups of elite endurance athletes and national endurance athletes (P=0.78), or between elite and national sprinters (P=0.46). In conclusion, the AMPD1 C34T mutation is not associated with endurance athletes’ status, at least in the present group of Israeli athletes. This suggests that elite athletic status can be achieved despite a partial deficiency in AMPD1, as marked by the AMPD1 CT genotype.

Casein Kinase 2 Regulates the mRNA-destabilizing Activity of Tristetraprolin

Tristetraprolin (TTP) is an AU-rich element-binding protein that regulates mRNA stability. We previously showed that TTP acts as a negative regulator of VEGF gene expression in colon cancer cells. The p38 MAPK pathway is known to suppress the TTP activity. However, until now the signaling pathway to enhance TTP function is not well known. Here, we show that casein kinase 2 (CK2) enhances the TTP function in the regulation of the VEGF expression in colon cancer cells. CK2 increased TTP protein levels and enhanced VEGF mRNA decaying activity of TTP. TTP was not a direct target of CK2. Instead, CK2 increased the phosphorylation of MKP-1, which led to a decrease in the phosphorylation of p38 MAPK. Inhibition of MKP-1 by siRNA attenuated the increase in TTP function and the decrease of p38 phosphorylation induced by CK2α overexpression. TGF-β1 increased the expressions of CK2 and TTP and the TTP function. The siRNA against CK2α or TTP reversed TGF-β1-induced increases in the expression of CK2 and TTP and the TTP function. Our data suggest that CK2 enhances the protein level and activity of TTP via the modulation of the MKP-1-p38 MAPK signaling pathway and that TGF-β1 enhances the activity of CK2.

RIP Pulchellin Isoforms: Biomembrane Models Showing Different Intoxication Mechanisms Between Them

Pulchellin, a type-2 Ribosome Inactivating Protein (RIP), whose action mechanism proposed would be initiate by B-chain binding on glycoconjugate onto the cell surface (lectinic chain). Into the cell, the A-chain (toxic chain) binding in ER lipids and stopping protein synthesis at ribosome inducing cell death. Was showed that Puchelin isoforms have different toxicity and were separated subgroups PI, PII, PIII, PIV based: toxicity, secondary structure, specificity for sugar binding. PII and PIV subgroups present structural identity above 70%, however PII is five-fold more toxic than PIV (Castilho, et al. FEBS journal, 2008). The present studies show results of interaction between model membranes and the two isoforms (PII and PIV) using Langmuir monolayers (LM) and giant unilamellar vesicles (GUVs). PII and PIV bond to LM-DPPC in the same extension, but when monolayers contented 5% of GalCer, PII remained strongly interacting with the mixed monolayer and PIV seems to be inhibited by the presence of the glycolipids. In GUVs-POPC, both PII and PIV destroyed the vesicles, however, in GUVs-POPC+5%GalCer, only PII induced vesicles decreased in their surface around 30% and some invaginations could be observed. After 20 minutes, small vesicles were observed inside of the GUV structure. For PIV, instead of invaginations, only fluctuations on the GUV surface were verified. Increasing PIV concentration the GUVs were destruction. In view of the results, one can conclude that the key for understanding differences in pulchellin isoforms toxicity is the interaction between B-chain lectinic and the glycosyl residues on cell surface. Supported by Brazilian Agencies: FAPESP, CNPq

New Selectable Marker for Manipulating the Simple Genomes of Mycoplasma Species

Over the past several years, significant advances have been made in the molecular genetics of the Mollicutes (the simplest cells that can be grown in axenic culture). Nevertheless, a number of basic molecular tools are still required before genetic manipulations become routine. Here we describe the development of a new dominant selectable marker based on the enzyme puromycin-N-acetyltransferase from Streptomyces alboniger. Puromycin is an antibiotic that mimics the 3'-terminal end of aminoacylated tRNAs and attaches to the carboxyl terminus of growing protein chains. This stops protein synthesis. Because puromycin conscripts rRNA recognition elements that are used by all of the various tRNAs in a cell, it is unlikely that spontaneous antibiotic resistance can be acquired via a simple point mutation--an annoying issue with existing mycoplasma markers. Our codon-optimized cassette confers pronounced puromycin resistance on all five of the mycoplasma species we have tested so far. The resistance cassette was also designed to function in Escherichia coli, which simplifies the construction of shuttle vectors and makes it trivial to produce the large quantities of DNA generally necessary for mycoplasma transformation. Due to these and other features, we expect the puromycin marker to be a widely applicable tool for studying these simple cells and pathogens.

A Phosphorylation Cascade Controls the Degradation of Active SREBP1

Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulates cholesterol and lipid metabolism. The active forms of these transcription factors are targeted by a number of post-translational modifications, including phosphorylation. Phosphorylation of Thr-426 and Ser-430 in SREBP1a creates a docking site for the ubiquitin ligase Fbw7, resulting in the degradation of the transcription factor. Here, we identify a novel phosphorylation site in SREBP1a, Ser-434, which regulates the Fbw7-dependent degradation of SREBP1. We demonstrate that both SREBP1a and SREBP1c are phosphorylated on this residue (Ser-410 in SREBP1c). Importantly, we demonstrate that the mature form of endogenous SREBP1 is phosphorylated on Ser-434. Glycogen synthase kinase-3 phosphorylates Ser-434, and the phosphorylation of this residue is attenuated in response to insulin signaling. Interestingly, phosphorylation of Ser-434 promotes the glycogen synthase kinase-3-dependent phosphorylation of Thr-426 and Ser-430 and destabilizes SREBP1. Consequently, mutation of Ser-434 blocks the interaction between SREBP1 and Fbw7 and attenuates Fbw7-dependent degradation of SREBP1. Importantly, insulin fails to enhance the levels of mature SREBP1 in cells lacking Fbw7. Thus, the degradation of mature SREBP1 is controlled by cross-talk between multiple phosphorylated residues in its C-terminal domain and the phosphorylation of Ser-434 could function as a molecular switch to control these processes.

Autolysis control hypotheses for tolerance to wall antibiotics.

Antibiotics are divided into two classes: bacteriostatic and bactericidal. The reasons for their classification are usually self-evident, although seldom proven. For example, chloramphenicol stops protein synthesis and is bacteriostatic, probably because when it is removed growth can recommence. Penicillin and congeners are bactericidal, probably because continued cytoplasmic synthesis should lead to increased cellular pressure and eventually to the rupture of the wall and to cell death. Lethality is not always the outcome, and certain strains may neither grow nor lyse in the presence of lactams (46–51, 55, 57, 60); they are called “tolerant.”

Optimization of a Yeast Estrogen Screen and Its Applicability to Study the Release of Estrogenic Isoflavones from a Soygerm Powder

Here we describe a redesigned protocol of the yeast estrogen screen developed by Routledge and Sumpter. The redesigned test comprises two steps. First, a large amount of yeast with estrogenic compounds is incubated for 24 hr. Subsequently, a mixture of cycloheximide and the chromogenic substrate chlorophenol red-beta-d-galactopyranoside (CPRG) is added. The cycloheximide stops protein synthesis and allows for an end-point measurement of beta-galactosidase activity generated during the first 24 hr. CPRG is converted to chlorophenol red and reflects beta-galactosidase activity, which is indicative of the estrogenic activity. The modifications shorten the duration of the assay at least 1 day and avoid interference of the estrogenic CPRG or chlorophenol red. The redesigned and the original protocol were used to study the estrogenic activity of bisphenol A, methoxychlor, p,p'-DDT, and isoflavones (genistein, daidzein, and glycitein). Bisphenol A, methoxychlor, and genistein triggered higher levels of beta-galactosidase activity in the redesigned protocol. Estrogenic activity of p,p'-DDT could only be demonstrated with the redesigned protocol. Glycitein and daidzein failed to give a response with both protocols. We also studied deconjugation of beta-glycosidic isoflavones present in soygerm powder. Treatment of the soygerm powder with beta-glycosidase released isoflavones. The estrogenic response of the samples was confirmed with the redesigned protocol and correlated with the amount of genistein present. The release of isoflavones under conditions prevailing in the intestines was studied. Bacterial beta-glycosidase present in the large intestine released isoflavones, and moderate estrogenic activity could be demonstrated.

Optimization of a yeast estrogen screen and its applicability to study the release of estrogenic isoflavones from a soygerm powder.

Here we describe a redesigned protocol of the yeast estrogen screen developed by Routledge and Sumpter. The redesigned test comprises two steps. First, a large amount of yeast with estrogenic compounds is incubated for 24 hr. Subsequently, a mixture of cycloheximide and the chromogenic substrate chlorophenol red-beta-d-galactopyranoside (CPRG) is added. The cycloheximide stops protein synthesis and allows for an end-point measurement of beta-galactosidase activity generated during the first 24 hr. CPRG is converted to chlorophenol red and reflects beta-galactosidase activity, which is indicative of the estrogenic activity. The modifications shorten the duration of the assay at least 1 day and avoid interference of the estrogenic CPRG or chlorophenol red. The redesigned and the original protocol were used to study the estrogenic activity of bisphenol A, methoxychlor, p,p'-DDT, and isoflavones (genistein, daidzein, and glycitein). Bisphenol A, methoxychlor, and genistein triggered higher levels of beta-galactosidase activity in the redesigned protocol. Estrogenic activity of p,p'-DDT could only be demonstrated with the redesigned protocol. Glycitein and daidzein failed to give a response with both protocols. We also studied deconjugation of beta-glycosidic isoflavones present in soygerm powder. Treatment of the soygerm powder with beta-glycosidase released isoflavones. The estrogenic response of the samples was confirmed with the redesigned protocol and correlated with the amount of genistein present. The release of isoflavones under conditions prevailing in the intestines was studied. Bacterial beta-glycosidase present in the large intestine released isoflavones, and moderate estrogenic activity could be demonstrated.

A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops

Colicin D has long been thought to stop protein synthesis in infected Escherichia coli cells by inactivating ribosomes, just like colicin E3. Here, we show that colicin D specifically cleaves tRNAs(Arg) including four isoaccepting molecules both in vivo and in vitro. The cleavage occurs in vitro between positions 38 and 39 in an anticodon loop with a 2',3'-cyclic phosphate end, and is inhibited by a specific immunity protein. Consistent with the cleavage of tRNAs(Arg), the RNA fraction of colicin-treated cells significantly reduced the amino acid-accepting activity only for arginine. Furthermore, we generated a single mutation of histidine in the C-terminal possible catalytic domain, which caused the loss of the killing activity in vivo together with the tRNA(Arg)-cleaving activity both in vivo and in vitro. These findings show that colicin D directly cleaves cytoplasmic tRNAs(Arg), which leads to impairment of protein synthesis and cell death. Recently, we found that colicin E5 stops protein synthesis by cleaving the anticodons of specific tRNAs for Tyr, His, Asn, and Asp. Despite these apparently similar actions on tRNAs and cells, colicins D and E5 not only exhibit no sequence homology but also have different molecular mechanisms as to both substrate recognition and catalytic reaction.

Glycerol Increases Content and Activity of Human Cytochrome P-4502E1 in a Transduced HepG2 Cell Line by Protein Stabilization

Glycerol is widely used to stabilize cytochrome P-450 and prevent its transformation to cytochrome P-420. The effect of glycerol on the content and activity of human cytochrome P-4502E1 (CYP2E1) in a HepG2 cell line that stably and constitutively expresses this P-450 was evaluated by immunoassays and oxidation of p-nitrophenol. Addition of 100 to 200 mM glycerol to the culture medium resulted in a 2 1/2- to 3-fold increase in the content and activity of CYP2E1 in microsomes isolated from the cells. Increases could be observed within 4 to 8 hr after addition of glycerol to the culture medium. Glycerol had no effect on the content of cytochrome b5 or activities of NADPH-cytochrome P-450 reductase or NADH-cytochrome b5 reductase. Upon the addition of cycloheximide to stop protein synthesis, CYP2E1 content and activity decreased with apparent half-lives of 6 and 4 hr, respectively. Glycerol prevented or decreased this loss of CYP2E1 content and activity. Labeling CYP2E1 with [35S]methionine, followed by pulse-chase experiments with cold methionine and immunoprecipitation of CYP2E1 indicated a half-life for CYP2E1 of approximately 3 hr. Glycerol increased the half-life to approximately 11 hr. Stabilization of CYP2E1 protein by glycerol was not additive or synergistic with the increase of CYP2E1 by ethanol or 4-methylpyrazole, suggesting that all three agents elevate CYP2E1 by a similar type of mechanism in this model. These results indicate that glycerol can interact with human CYP2E1 to stabilize it against proteolytic degradation, increasing the half-life of the enzyme and thereby elevating the content and activity of CYP2E1.