Tigliane-Type Diterpene Esters from the Fruits of Shirakiopsis indica and Their Anti-HIV Activity.

Four new diterpene esters, shirakindicans A-D (1-4), along with eight related known diterpene esters (5-12), were isolated from the fruits of the Bangladeshi medicinal plant Shirakiopsis indica. The structures of 1-4 were elucidated by spectroscopic data analysis and electronic circular dichroism (ECD) calculations. Shirakindican A (1) was assigned as a tigliane-type diterpene ester possessing an unusual 6ß-hydroxy-1,7-dien-3-one structure, while shirakindican B (2) exhibits a tiglia-1,5-dien-3,7-dione structure. The anti-HIV activities of the isolated diterpene esters were evaluated and showed significant activities for sapintoxins A (5) and D (11), with EC50 values of 0.0074 and 0.044 µM, respectively, and TI values of 1 100 and 5 290. Sapatoxin A (12) also exhibited anti-HIV activity with an EC50 value of 0.13 µM and a TI value of 161.

Air Quality Dispersion Modelling to Evaluate CIPP Installation Styrene Emissions.

Cured-in-place pipe (CIPP) is one of the most popular in situ rehabilitation techniques to repair sewer and water pipes. While there are multiple approaches to curing CIPP, steam-curing of styrene-based resins has been found to be associated with air-borne chemical emissions. Health officials, utilities and industry representatives have recognized the need to know more about these emissions, especially styrene. Such concern has led to multiple studies investigating the concentrations of volatile organic compounds on CIPP installation sites. This study expands upon previous effort by modeling worst-case, steam-cured CIPP emissions over a 5-year weather record. The effort also includes calibration of the model to emissions averages over the work day rather than instantaneous field measurements. Dispersion modelling software, AERMOD, was utilized to model the styrene component of CIPP emissions on two CIPP installation sites in the US. Based on the analysis results, it was found that the styrene emitted from stacks dissipates rapidly with styrene concentrations only exceeding minimum health and safety threshold levels at distances close to the stack (2 m or less). The values predicted by the model analysis are comparable with the field measured styrene concentrations from other studies. Current safety guidelines in the US recommend a 4.6-m (15-ft) safety perimeter for stack emission points. The results of this study indicate that significant and lasting health impacts are unlikely outside recommended safety perimeter. The results also validate the importance of enforcing recommended safety guidance on steam-cured CIPP sites.

Genetic Biosensor Design for Natural Product Biosynthesis in Microorganisms.

Low yield and low titer of natural products are common issues in natural product biosynthesis through microbial cell factories. One effective way to resolve such bottlenecks is to design genetic biosensors to monitor and regulate the biosynthesis of target natural products. In this review, we evaluate the most recent advances in the design of genetic biosensors for natural product biosynthesis in microorganisms. In particular, we examine strategies for selection of genetic parts and construction principles for the design and evaluation of genetic biosensors. We also review the latest applications of transcription factor- and riboswitch-based genetic biosensors in natural product biosynthesis. Lastly, we discuss challenges and solutions in designing genetic biosensors for the biosynthesis of natural products in microorganisms.

The Potential for microRNA Therapeutics and Clinical Research.

As FDA-approved small RNA drugs start to enter clinical medicine, ongoing studies for the microRNA (miRNA) class of small RNAs expand its preclinical and clinical research applications. A growing number of reports suggest a significant utility of miRNAs as biomarkers for pathogenic conditions, modulators of drug resistance, and/or as drugs for medical intervention in almost all human health conditions. The pleiotropic nature of this class of nonprotein-coding RNAs makes them particularly attractive drug targets for diseases with a multifactorial origin and no current effective treatments. As candidate miRNAs begin to proceed toward initiation and completion of potential phase 3 and 4 trials in the future, the landscape of both diagnostic and interventional medicine will arguably continue to evolve. In this mini-review, we discuss miRNA drug discovery development and their current status in clinical trials.

Rewriting the Metabolic Blueprint: Advances in Pathway Diversification in Microorganisms.

Living organisms have evolved over millions of years to fine tune their metabolism to create efficient pathways for producing metabolites necessary for their survival. Advancement in the field of synthetic biology has enabled the exploitation of these metabolic pathways for the production of desired compounds by creating microbial cell factories through metabolic engineering, thus providing sustainable routes to obtain value-added chemicals. Following the past success in metabolic engineering, there is increasing interest in diversifying natural metabolic pathways to construct non-natural biosynthesis routes, thereby creating possibilities for producing novel valuable compounds that are non-natural or without elucidated biosynthesis pathways. Thus, the range of chemicals that can be produced by biological systems can be expanded to meet the demands of industries for compounds such as plastic precursors and new antibiotics, most of which can only be obtained through chemical synthesis currently. Herein, we review and discuss novel strategies that have been developed to rewrite natural metabolic blueprints in a bid to broaden the chemical repertoire achievable in microorganisms. This review aims to provide insights on recent approaches taken to open new avenues for achieving biochemical production that are beyond currently available inventions.

Metabolic engineering of cofactor flavin adenine dinucleotide (FAD) synthesis and regeneration in Escherichia coli for production of α-keto acids.

Cofactor flavin adenine dinucleotide (FAD) plays a vital role in many FAD-dependent enzymatic reactions; therefore, how to efficiently accelerate FAD synthesis and regeneration is an important topic in biocatalysis and metabolic engineering. In this study, a system involving the synthesis pathway and regeneration of FAD was engineered in Escherichia coli to improve α-keto acid production-from the corresponding l-amino acids-catalyzed by FAD-dependent l-amino acid deaminase (l-AAD). First, key genes, ribH, ribC, and ribF, were overexpressed and fine-tuned for FAD synthesis. In the resulting E. coli strain PHCF7, strong overexpression of pma, ribC, and ribF and moderate overexpression of ribH yielded a 90% increase in phenylpyruvic acid (PPA) titer: 19.4 ± 1.1 g · L-1 . Next, formate dehydrogenase (FDH) and NADH oxidase (NOX) were overexpressed to strengthen the regeneration rate of cofactors FADH2 /FAD using FDH for FADH2 /FAD regeneration and NOX for NAD+ /NADH regeneration. The resulting E. coli strain PHCF7-FDH-NOX yielded the highest PPA production: 31.4 ± 1.1 g · L-1 . Finally, this whole-cell system was adapted to production of other α-keto acids including α-ketoglutaric acid, α-ketoisocaproate, and keto-γ-methylthiobutyric acid to demonstrate the broad utility of strengthening of FAD synthesis and FADH2 /FAD regeneration for production of α-keto acids. Notably, the strategy reported herein may be generally applicable to other flavin-dependent biocatalysis reactions and metabolic pathway optimizations. Biotechnol. Bioeng. 2017;114: 1928-1936. © 2017 Wiley Periodicals, Inc.

Metabolic Engineering of Raoultella ornithinolytica BF60 for Production of 2,5-Furandicarboxylic Acid from 5-Hydroxymethylfurfural.

2,5-Furandicarboxylic acid (FDCA) is an important renewable biotechnological building block because it serves as an environmentally friendly substitute for terephthalic acid in the production of polyesters. Currently, FDCA is produced mainly via chemical oxidation, which can cause severe environmental pollution. In this study, we developed an environmentally friendly process for the production of FDCA from 5-hydroxymethyl furfural (5-HMF) using a newly isolated strain, Raoultella ornithinolytica BF60. First, R. ornithinolytica BF60 was identified by screening and was isolated. Its maximal FDCA titer was 7.9 g/liter, and the maximal molar conversion ratio of 5-HMF to FDCA was 51.0% (mol/mol) under optimal conditions (100 mM 5-HMF, 45 g/liter whole-cell biocatalyst, 30°C, and 50 mM phosphate buffer [pH 8.0]). Next, dcaD, encoding dicarboxylic acid decarboxylase, was mutated to block FDCA degradation to furoic acid, thus increasing FDCA production to 9.2 g/liter. Subsequently, aldR, encoding aldehyde reductase, was mutated to prevent the catabolism of 5-HMF to HMF alcohol, further increasing the FDCA titer, to 11.3 g/liter. Finally, the gene encoding aldehyde dehydrogenase 1 was overexpressed. The FDCA titer increased to 13.9 g/liter, 1.7 times that of the wild-type strain, and the molar conversion ratio increased to 89.0%. IMPORTANCE: In this work, we developed an ecofriendly bioprocess for green production of FDCA in engineered R. ornithinolytica This report provides a starting point for further metabolic engineering aimed at a process for industrial production of FDCA using R. ornithinolytica.

Two-Step Production of Phenylpyruvic Acid from L-Phenylalanine by Growing and Resting Cells of Engineered Escherichia coli: Process Optimization and Kinetics Modeling.

Phenylpyruvic acid (PPA) is widely used in the pharmaceutical, food, and chemical industries. Here, a two-step bioconversion process, involving growing and resting cells, was established to produce PPA from l-phenylalanine using the engineered Escherichia coli constructed previously. First, the biotransformation conditions for growing cells were optimized (l-phenylalanine concentration 20.0 g·L-1, temperature 35°C) and a two-stage temperature control strategy (keep 20°C for 12 h and increase the temperature to 35°C until the end of biotransformation) was performed. The biotransformation conditions for resting cells were then optimized in 3-L bioreactor and the optimized conditions were as follows: agitation speed 500 rpm, aeration rate 1.5 vvm, and l-phenylalanine concentration 30 g·L-1. The total maximal production (mass conversion rate) reached 29.8 ± 2.1 g·L-1 (99.3%) and 75.1 ± 2.5 g·L-1 (93.9%) in the flask and 3-L bioreactor, respectively. Finally, a kinetic model was established, and it was revealed that the substrate and product inhibition were the main limiting factors for resting cell biotransformation.

Metabolic engineering for amino-, oligo-, and polysugar production in microbes.

Amino-, oligo-, and polysugars are important for both medicinal and industrial applications. Microbial processes used in production of such sugars are not only carbon-intensive and energy-demanding processes but also have other distinct disadvantages such as low productivity, low yields, and by-product contamination. Therefore, metabolic engineering has emerged as an effective tool for developing engineered strains to deliver production strategies for many valuable sugars, which were previously difficult to manufacture by other means, in necessary amounts to support their applications. In this review, the recent strategies used for metabolic engineering are summarized and future prospects of this technique are discussed. We hope that this review will contribute to the development of functional and high-value sugar production by metabolic engineering strategies.

Combination of phenylpyruvic acid (PPA) pathway engineering and molecular engineering of L-amino acid deaminase improves PPA production with an Escherichia coli whole-cell biocatalyst.

In our previous study, we produced phenylpyruvic acid (PPA) in one step from L-phenylalanine by using an Escherichia coli whole-cell biocatalyst expressing an L-amino acid deaminase (L-AAD) from Proteus mirabilis KCTC2566. However, the PPA titer was low due to the degradation of PPA and low substrate specificity of L-AAD. In this study, metabolic engineering of the L-phenylalanine degradation pathway in E. coli and protein engineering of L-AAD from P. mirabilis were performed to improve the PPA titer. First, three aminotransferase genes were knocked out to block PPA degradation, which increased the PPA titer from 3.3 ± 0.2 to 3.9 ± 0.1 g/L and the substrate conversion ratio to 97.5 %. Next, L-AAD was engineered via error-prone polymerase chain reaction, followed by site-saturation mutation to improve its catalytic performance. The triple mutant D165K/F263M/L336M produced the highest PPA titer of 10.0 ± 0.4 g/L, with a substrate conversion ratio of 100 %, which was 3.0 times that of wild-type L-AAD. Comparative kinetics analysis showed that compared with wild-type L-AAD, the triple mutant had higher substrate-binding affinity and catalytic efficiency. Finally, an optimal fed-batch biotransformation process was developed to achieve a maximal PPA titer of 21 ± 1.8 g/L within 8 h. This study developed a robust whole-cell E. coli biocatalyst for PPA production by integrating metabolic and protein engineering, strategies that may be useful for the construction of other biotransformation biocatalysts.

Production of phenylpyruvic acid from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis: comparison of enzymatic and whole-cell biotransformation approaches.

Phenylpyruvic acid (PPA) is an important organic acid that has a wide range of applications. In this study, the membrane-bound L-amino acid deaminase (L-AAD) gene from Proteus mirabilis KCTC 2566 was expressed in Escherichia coli BL21(DE3) and then the L-AAD was purified. After that, we used the purified enzyme and the recombinant E. coli whole-cell biocatalyst to produce PPA via a one-step biotransformation from L-phenylalanine. L-AAD was solubilized from the membrane and purified 52-fold with an overall yield of 13 %, which corresponded to a specific activity of 0.94 ± 0.01 µmol PPA min(-1)·mg(-1). Then, the biotransformation conditions for the pure enzyme and the whole-cell biocatalyst were optimized. The maximal production was 2.6 ± 0.1 g·L(-1) (specific activity of 1.02 ± 0.02 µmol PPA min(-1)·mg(-1) protein, 86.7 ± 5 % mass conversion rate, and 1.04 g·L(-1)·h(-1) productivity) and 3.3 ± 0.2 g L(-1) (specific activity of 0.013 ± 0.003 µmol PPA min(-1)·mg(-1) protein, 82.5 ± 4 % mass conversion rate, and 0.55 g·L(-1)·h(-1) productivity) for the pure enzyme and whole-cell biocatalyst, respectively. Comparative studies of the enzymatic and whole-cell biotransformation were performed in terms of specific activity, production, conversion, productivity, stability, need of external cofactors, and recycling. We have developed two eco-friendly and efficient approaches for PPA production. The strategy described herein may aid the biotransformational synthesis of other α-keto acids from their corresponding amino acids.

One-step biosynthesis of α-keto-γ-methylthiobutyric acid from L-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered L-amino acid deaminase from Proteus vulgaris.

α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.

Improved production of α-ketoglutaric acid (α-KG) by a Bacillus subtilis whole-cell biocatalyst via engineering of L-amino acid deaminase and deletion of the α-KG utilization pathway.

We previously developed a novel one-step biotransformation process for the production of α-ketoglutarate (α-KG) from L-glutamic acid by a Bacillus subtilis whole-cell biocatalyst expressing an L-amino acid deaminase (pm1) of Proteus mirabilis. However, the biotransformation efficiency of this process was low owing to low substrate specificity and high α-KG degradation. In this study, we further improved α-KG production by protein engineering P. mirabilis pm1 and deleting the B. subtilis α-KG degradation pathway. We first performed three rounds of error-prone polymerase chain reaction and identified mutations at six sites (F110, A255, E349, R228, T249, and I352) that influence catalytic efficiency. We then performed site-saturation mutagenesis at these sites, and the mutant F110I/A255T/E349D/R228C/T249S/I352A increased the biotransformation ratio of L-glutamic acid from 31% to 83.25% and the α-KG titer from 4.65 g/L to 10.08 g/L. Next, the reaction kinetics and biochemical properties of the mutant were analyzed. The Michaelis constant for L-glutamic acid decreased from 49.21 mM to 23.58 mM, and the maximum rate of α-KG production increased from 22.82 µM min(-1) to 56.7 µM min(-1). Finally, the sucA gene, encoding α-ketodehydrogenase, was deleted to reduce α-KG degradation, increasing the α-KG titer from 10.08 g/L to 12.21 g/L. Protein engineering of P. mirabilis pm1 and deletion of the α-KG degradation pathway in B. subtilis improved α-KG production over that of previously developed processes.

L-Amino acid oxidases from microbial sources: types, properties, functions, and applications.

L-Amino acid oxidases (LAAOs), which catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids and ammonia, are flavin adenine dinucleotide-containing homodimeric proteins. L-Amino acid oxidases are widely distributed in diverse organisms and have a range of properties. Because expressing LAAOs as recombinant proteins in heterologous hosts is difficult, their biotechnological applications have not been thoroughly advanced. LAAOs are thought to contribute to amino acid catabolism, enhance iron acquisition, display antimicrobial activity, and catalyze keto acid production, among other roles. Here, we review the types, properties, structures, biological functions, heterologous expression, and applications of LAAOs obtained from microbial sources. We expect this review to increase interest in LAAO studies.

Bioconversion of l-glutamic acid to α-ketoglutaric acid by an immobilized whole-cell biocatalyst expressing l-amino acid deaminase from Proteus mirabilis.

The goal of this work was to develop an immobilized whole-cell biocatalytic process for the environment-friendly synthesis of α-ketoglutaric acid (α-KG) from l-glutamic acid. We compared the suitability of Escherichia coli and Bacillus subtilis strains overexpressing Proteus mirabilisl-amino acid deaminase (l-AAD) as potential biocatalysts. Although both recombinant strains were biocatalytically active, the performance of B. subtilis was superior to that of E. coli. With l-glutamic acid as the substrate, α-KG production levels by membranes isolated from B. subtilis and E. coli were 55.3±1.73 and 21.7±0.39µg/mg protein/min, respectively. The maximal conversion ratio of l-glutamic acid to α-KG was 31% (w/w) under the following optimal conditions: 15g/L l-glutamic acid, 20g/L whole-cell biocatalyst, 5mM MgCl2, 40°C, pH 8.0, and 24-h incubation. Immobilization of whole cells with alginate increased the recyclability by an average of 23.33% per cycle. This work established an efficient one-step biotransformation process for the production of α-KG using immobilized whole B. subtilis overexpressing P. mirabilisl-AAD. Compared with traditional multistep chemical synthesis, the biocatalytic process described here has the advantage of reducing environmental pollution and thus has great potential for the large-scale production of α-KG.

Deficiency of TDAG51 protects against atherosclerosis by modulating apoptosis, cholesterol efflux, and peroxiredoxin-1 expression.

BACKGROUND: Apoptosis caused by endoplasmic reticulum (ER) stress contributes to atherothrombosis, the underlying cause of cardiovascular disease (CVD). T-cell death-associated gene 51 (TDAG51), a member of the pleckstrin homology-like domain gene family, is induced by ER stress, causes apoptosis when overexpressed, and is present in lesion-resident macrophages and endothelial cells. METHODS AND RESULTS: To study the role of TDAG51 in atherosclerosis, male mice deficient in TDAG51 and apolipoprotein E (TDAG51(-/-)/ApoE(-/-)) were generated and showed reduced atherosclerotic lesion growth (56 ± 5% reduction at 40 weeks, relative to ApoE(-/-) controls, P<0.005) and necrosis (41 ± 4% versus 63 ± 8% lesion area in TDAG51(-/-)/ApoE(-/-) and ApoE(-/-), respectively; P<0.05) without changes in plasma levels of lipids, glucose, and inflammatory cytokines. TDAG51 deficiency caused several phenotypic changes in macrophages and endothelial cells that increase cytoprotection against oxidative and ER stress, enhance PPARγ-dependent reverse cholesterol transport, and upregulate peroxiredoxin-1 (Prdx-1), an antioxidant enzyme with antiatherogenic properties (1.8 ± 0.1-fold increase in Prdx-1 protein expression, relative to control macrophages; P<0.005). Two independent case-control studies found that a genetic variant in the human TDAG51 gene region (rs2367446) is associated with CVD (OR, 1.15; 95% CI, 1.07 to 1.24; P=0.0003). CONCLUSIONS: These findings provide evidence that TDAG51 affects specific cellular pathways known to reduce atherogenesis, suggesting that modulation of TDAG51 expression or its activity may have therapeutic benefit for the treatment of CVD.

One-step production of α-ketoglutaric acid from glutamic acid with an engineered L-amino acid deaminase from Proteus mirabilis.

Currently, α-ketoglutaric acid (α-KG) is industrially produced by multi-step chemical synthesis, which can cause heavy environmental pollution. Here we reported a simple one-step approach for the production of α-KG by transforming l-glutamic acid with an engineered l-amino acid deaminase (l-AAD) from Proteus mirabilis. First, to facilitate the purification of membrane-bound l-AAD, one N-terminal transmembrane region (from 21 to 87th nucleotide) was removed from l-AAD to block the binding of l-AAD with membrane, and the relatively low-usage codons were replaced by high-usage codons in Escherichia coli to improve the expression level. However, inclusion bodies formed when expressing the ΔN-LAAD in E. coli BL 21, and then the soluble and active ΔN-LAAD was obtained by the solubilization and renaturation of ΔN-LAAD. Furthermore, the biochemical properties of the refolded ΔN-LAAD were characterized and compared with those of full-length l-AAD. Finally, the ΔN-LAAD was used to synthesize α-KG and the maximal formation rate of α-KG reached 12.6% (w/w) at 6h under the following conditions: 12g/L l-glutamic acid, 0.1g/L ΔN-LAAD, 5mM MgCl2, temperature 45°C and pH 8.0. Compared with the multi-step chemical synthesis, the transformation approach has less environmental pollution and has a great potential for α-KG production.

Prevalence of biliary ascariasis and its relation to biliary lithiasis.

Hepatobiliary ultrasound was carried out on 2224 consecutive patients at the Center for Nuclear Medicine and Ultrasound and at one private diagnostic center in Mymensingh for ultrasound examination of different systems. The purpose of this study was to investigate the prevalence of biliary ascariasis and its association with other biliary diseases, specially biliary lithiasis. Data regarding the presence of stone, worm or other diseases were recorded accordingly in this prospective study. There were 952 male and 1272 female patients with an age range of 5-90 years. Biliary diseases were detected in 305 patients (13.71%), of whom 97 were male (10.19%) and 208 were female (16.35%). The most common biliary disease in both sexes was cholelithiasis (11.87%), which was found in 84 male patients (8.82%) and in 180 female patients (14.15%). Other diseases found were choledocholithiasis in 14 patients (3 males, 11 females), gall bladder mass in 9 patients (3 males, 6 females), common bile duct mass in 7 patients (4 male, 3 female) and biliary ascariasis in 10 patients (3 male, 7 female). Overall prevalence of biliary ascariasis was 0.45% (0.31% in male patients and 0.55% in female patients), and age range of patients with the condition was 6-50 years. No case of biliary ascariasis was associated with cholelithiasis or choledocholithiasis. Acute cholecystitis was associated with 8 cases (80%) of biliary ascariasis. Common sonographic findings in patients with biliary ascariasis were a single long, linear or curved echogenic structure within the bile duct, without acoustic shadowing. Other findings were gall bladder distention with sludge inside, an edematous wall and mildly dilated biliary tree. Prevalence of biliary ascariasis in the study was 0.45%, with incidence being higher in female subjects (0.55%). No correlation was found between biliary ascariasis and biliary lithiasis. Most of the cases of biliary ascariasis were associated with acute cholecystitis. We concluded that a careful search for biliary ascariasis should be considered for patients with acute acalculus cholecystitis, especially in areas in which ascariasis is endemic, such as Bangladesh.

Peroxynitrite causes endoplasmic reticulum stress and apoptosis in human vascular endothelium: implications in atherogenesis.

OBJECTIVE: Peroxynitrite, a potent oxidant generated by the reaction of NO with superoxide, has been implicated in the promotion of atherosclerosis. We designed this study to determine whether peroxynitrite induces its proatherogenic effects through induction of endoplasmic reticulum (ER) stress. METHODS AND RESULTS: Human vascular endothelial cells treated with Sin-1, a peroxynitrite generator, induced the expression of the ER chaperones GRP78 and GRP94 and increased eIF2alpha phosphorylation. These effects were inhibited by the peroxynitrite scavenger uric acid. Sin-1 caused the depletion of ER-Ca2+, an effect known to induce ER stress, resulting in the elevation of cytosolic Ca2+ and programmed cell death (PCD). Sin-1 treatment was also found, via 3-nitrotyrosine and GRP78 colocalization, to act directly on the ER. Adenoviral-mediated overexpression of GRP78 in endothelial cells prevented Sin-1-induced PCD. Consistent with these in vitro findings, 3-nitrotyrosine was observed and colocalized with GRP78 in endothelial cells of early atherosclerotic lesions from apolipoprotein E-deficient mice. CONCLUSIONS: Peroxynitrite is an ER stress-inducing agent. Its effects include the depletion of ER-Ca2+, a known mechanism of ER stress induction. The observation that 3-nitrotyrosine-containing proteins colocalize with markers of ER stress within early atherosclerotic lesions suggests that peroxynitrite contributes to atherogenesis through a mechanism involving ER stress.

Association of multiple cellular stress pathways with accelerated atherosclerosis in hyperhomocysteinemic apolipoprotein E-deficient mice.

BACKGROUND: A causal relation between hyperhomocysteinemia (HHcy) and accelerated atherosclerosis has been established in apolipoprotein E-deficient (apoE-/-) mice. Although several cellular stress mechanisms have been proposed to explain the atherogenic effects of HHcy, including oxidative stress, endoplasmic reticulum (ER) stress, and inflammation, their association with atherogenesis has not been completely elucidated. METHODS AND RESULTS: ApoE-/- mice were fed a control or a high-methionine (HM) diet for 4 (early lesion group) or 18 (advanced lesion group) weeks to induce HHcy. Total plasma homocysteine levels and atherosclerotic lesion size were significantly increased in early and advanced lesion groups fed the HM diet compared with control groups. Markers of ER stress (GRP78/94, phospho-PERK), oxidative stress (HSP70), and inflammation (phospho-IkappaB-alpha) were assessed by immunohistochemical staining of these atherosclerotic lesions. GRP78/94, HSP70, and phospho-IkappaB-alpha immunostaining were significantly increased in the advanced lesion group fed the HM diet compared with the control group. HSP47, an ER-resident molecular chaperone involved in collagen folding and secretion, was also increased in advanced lesions of mice fed the HM diet. GRP78/94 and HSP47 were predominantly localized to the smooth muscle cell-rich fibrous cap, whereas HSP70 and phospho-IkappaB-alpha were observed in the lipid-rich necrotic core. Increased HSP70 and phospho-IkappaB-alpha immunostaining in advanced lesions of mice fed the HM diet are consistent with enhanced carotid artery dihydroethidium staining. Interestingly, GRP78/94 and phospho-PERK were markedly increased in macrophage foam cells from early lesions of mice fed the control or the HM diet. CONCLUSIONS: Multiple cellular stress pathways, including ER stress, are associated with atherosclerotic lesion development in apoE-/- mice.