Large-scale profiling of noncoding RNA function in yeast

Noncoding RNAs (ncRNAs) are emerging as key regulators of cellular function. We have exploited the recently developed barcoded ncRNA gene deletion strain collections in the yeast Saccharomyces cerevisiae to investigate the numerous ncRNAs in yeast with no known function. The ncRNA deletion collection contains deletions of tRNAs, snoRNAs, snRNAs, stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs) and other annotated ncRNAs encompassing 532 different individual ncRNA deletions. We have profiled the fitness of the diploid heterozygous ncRNA deletion strain collection in six conditions using batch and continuous liquid culture, as well as the haploid ncRNA deletion strain collections arrayed individually onto solid rich media. These analyses revealed many novel environmental-specific haplo-insufficient and haplo-proficient phenotypes providing key information on the importance of each specific ncRNA in every condition. Co-fitness analysis using fitness data from the heterozygous ncRNA deletion strain collection identified two ncRNA groups required for growth during heat stress and nutrient deprivation. The extensive fitness data for each ncRNA deletion strain has been compiled into an easy to navigate database called Yeast ncRNA Analysis (YNCA). By expanding the original ncRNA deletion strain collection we identified four novel essential ncRNAs; SUT527, SUT075, SUT367 and SUT259/691. We defined the effects of each new essential ncRNA on adjacent gene expression in the heterozygote background identifying both repression and induction of nearby genes. Additionally, we discovered a function for SUT527 in the expression, 3’ end formation and localization of SEC4, an essential protein coding mRNA. Finally, using plasmid complementation we rescued the SUT075 lethal phenotype revealing that this ncRNA acts in trans. Overall, our findings provide important new insights into the function of ncRNAs

Encapsulated or amphiphilic liposomal Fe(III) coordination complexes for MRI studies of tumor uptake and clearance

Liposomes containing high-spin Fe(III) coordination complexes were prepared towards the production of MRI probes with improved relaxivity and rapid pharmacokinetic clearance in mice. The amphiphilic Fe(III) complexes were anchored into the liposome with two alkyl chains to give a coordination sphere containing mixed amide hydroxypropyl pendant groups. Three types of MRI probes were prepared including those with intraliposomal Fe(III) complex (LipoA) alone, amphiphilic Fe(III) complex (LipoB) or both intraliposomal and amphiphilic complex (LipoC). Water proton relaxivities r1 and r2 were measured and compared to a small molecule macrocyclic Fe(III) complex containing similar donor groups. Liposomes with amphiphilic Fe(III) complex (LipoB) have a per particle relaxivity of 37,000 and a per iron relaxivity of 2.6 mM-1s-1 in solutions with pH 7.2, 34 C at 1.4 T. Liposomes containing both amphiphilic and intraliposomal Fe(III) complexes (lipoC) have reduced per iron relaxivity of 0.8 mM-1s-1 in solution consistent with quenching of the interior Fe(III) complex relaxivity and per particle relaxivity of 42 ,000 mM-1s-1. Liposomes containing only encapsulated Fe(III) complex have a lower relaxivity of 0.46 mM-1s-1 per iron complex. Studies show that lipoB and lipoC produce enhanced signal in the CT26 tumors of BALB/c mice. However, the biodistribution and clearance of the liposomal nanoparticles differs greatly. LipoB is a blood pool agent with a long circulation time whereas lipoC is cleared more rapidly through both renal and hepatobiliary pathways

Modulating the Properties of Fe(III) Macrocyclic MRI Contrast Agents by Appending Sulfonate or Hydroxyl Groups

Complexes of Fe(III) that contain a triazacyclononane (TACN) macrocycle, two pendant hydroxyl groups, and a third ancillary pendant show promise as MRI contrast agents. The ancillary group plays an important role in tuning the solution relaxivity of the Fe(III) complex and leads to large changes in MRI contrast enhancement in mice. Two new Fe(III) complexes, one with a third coordinating hydroxypropyl pendant, Fe(L2), and one with an anionic non-coordinating sulfonate group, Fe(L1)(OH2), are compared. Both complexes have a deprotonated hydroxyl group at neutral pH and electrode potentials representative of a stabilized trivalent iron center. The r1 relaxivity of the Fe(L1)(OH2) complex is double that of the saturated complex, Fe(L2), at 4.7 T, 37 °C in buffered solutions. However, variable-temperature 17O-NMR experiments show that the inner-sphere water of Fe(L1)(OH2) does not exchange rapidly with bulk water under these conditions. The pendant sulfonate group in Fe(L1)(OH2) confers high solubility to the complex in comparison to Fe(L2) or previously studied analogues with benzyl groups. Dynamic MRI studies of the two complexes showed major differences in their pharmacokinetics clearance rates compared to an analogue containing a benzyl ancillary group. Rapid blood clearance and poor binding to serum albumin identify Fe(L1)(OH2) for development as an extracellular fluid contrast agent

TNF Signaling Is Required for Castration-Induced Vascular Damage Preceding Prostate Cancer Regression

The mainstay treatment for locally advanced, recurrent, or metastatic prostate cancer (PrCa) is androgen deprivation therapy (ADT). ADT causes prostate cancers to shrink in volume, or regress, by inducing epithelial tumor cell apoptosis. In normal, non-neoplastic murine prostate, androgen deprivation via castration induces prostate gland regression that is dependent on TNF signaling. In addition to this direct mechanism of action, castration has also been implicated in an indirect mechanism of prostate epithelial cell death, which has been described as vascular regression. The initiating event is endothelial cell apoptosis and/or increased vascular permeability. This subsequently leads to reduced blood flow and perfusion, and then hypoxia, which may enhance epithelial cell apoptosis. Castration-induced vascular regression has been observed in both normal and neoplastic prostates. We used photoacoustic, power Doppler, and contrast-enhanced ultrasound imaging, and CD31 immunohistochemical staining of the microvasculature to assess vascular integrity in the period immediately following castration, enabling us to test the role of TNF signaling in vascular regression. In two mouse models of androgen-responsive prostate cancer, TNF signaling blockade using a soluble TNFR2 ligand trap reversed the functional aspects of vascular regression as well as structural changes in the microvasculature, including reduced vessel wall thickness, cross-sectional area, and vessel perimeter length. These results demonstrate that TNF signaling is required for vascular regression, most likely by inducing endothelial cell apoptosis and increasing vessel permeability. Since TNF is also the critical death receptor ligand for prostate epithelial cells, we propose that TNF is a multi-purpose, comprehensive signal within the prostate cancer microenvironment that mediates prostate cancer regression following androgen deprivation

PROTIPIAR

Abstract. Tumor progression and angiogenesis are intimately related. To understand the interrelationship between these two processes, real-time imaging can make a major contribution. In this report, fluorescent protein imaging (FPI) and magnetic resonance imaging (MRI) were utilized to demonstrate the effects of selenium on tumor progression and angiogenesis in an orthotopic model of human colon cancer. GEO (welldifferentiated human colon carcinoma), cells transfected with green fluorescent protein (GFP), were implanted orthotopically into the colon of athymic nude mice. Beginning five days post implantation, whole-body FPI was performed to monitor tumor growth in vivo. Upon successful visualization of tumor growth by FPI, animals were randomly assigned to either a control group or a treatment group. Treatment consisted of daily oral administration of the organoselenium compound, methylselenocysteine (MSC; 0.2 mg/day × five weeks). Dynamic contrast-enhanced MRI was performed to examine the change in tumor blood volume following treatment. CD31 immunostaining of tumor sections was also performed to quantify microvessel density (MVD). While T1-and T2-weighted MRI provided adequate contrast and volumetric assessment of GEO tumor growth, GFP imaging allowed for high-throughput visualization of tumor progression in vivo. Selenium treatment resulted in a significant reduction in blood volume and microvessel density of GEO tumors. A significant inhibition of tumor growth was also observed in selenium-treated animals compared to untreated control animals. Together, these results highlight the usefulness of multimodal imaging approaches to demonstrate antitumor and anti-angiogenesis efficacy and the potential of selenium treatment of colon cancer. The development of targeted therapies for cancer in recent years has been accompanied by parallel advances in imaging technologies to enable assessment of their efficacy. In preclinical model systems, several imaging modalities including magnetic resonance imaging (MRI), computed tomography (CT), positron-emission tomography (PET) and optical imaging methods have been utilized to evaluate the pharmacologic activity of novel targeted therapies (1-3). However, each of these imaging methods has distinguishable advantages and limitations as well as marked differences in their sensitivity of detection, and spatial and temporal resolution. The use of multiple imaging methods is therefore likely to provide complimentary information on tumor biology. Studies have previously examined the use of optical imaging techniques such as fluorescent protein imaging (FPI) and bioluminescence imaging in combination with clinical imaging methods such as Materials and Methods Animals. Eight-to-twelve-week-old female athymic nude mice (nu/nu, body weight, 20-25 g) were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN, USA) and housed in microisolator cages (four to five animals per cage). Animals were provided with food and water ad libitum and maintained on 12-hour light/dark cycles in a HEPA-filtered environment within the Laboratory Animal Resource at Roswell Park Cancer Institute. Surgical procedure for orthotopic implantation. GEO human cancer cells were labeled with green fluorescent protein (GFP) using a retroviral transfection technique described previously (10). Subcutaneous tumors were initially established in donor mice by injection of 5×10 6 GEO cells under isoflurane anesthesia (Abbott Laboratories, Abbott Park, IL, USA). Upon successful establishment of tumors (~500 mm 3 ) from the cell lines for at least two passages, tumors were harvested and cut into smaller fragments (~1 mm/20 mg) for surgical orthotopic implantation into recipient mice. Under transient isoflurane anesthesia, a 1 cm laparotomy was performed and two individual pieces of tumor tissue were subserosally implanted into the cecum and ascending colon using an aseptic technique (10-12). Surgical procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use committee at Roswell Park Cancer Institute. Fluorescence imaging. Fluorescence imaging was performed periodically to monitor tumor growth and progression in vivo. Images were obtained using a fluorescence imaging system (Light Box; Light Tools Research, Encinitas, CA, USA) equipped with a fiber optic illumination at 470 nm. Images were collected through a 515 nm long-pass filter and captured using Imaging software (Burnaby, British Columbia, Canada). Images were optimized for contrast and brightness using commercial software (Adobe Photoshop, CS2; Adobe, San Jose, CA, USA). Drug treatment. MSC was obtained from Sigma (St. Louis, MO, USA) and dissolved in sterile saline at a concentration of 1 mg/ml. For studies, MSC was administered orally at a dose of 0.2 mg/mouse/day beginning 6 days after tumor implantation. Magnetic resonance imaging. MRI studies were performed using a 4.7-T/33-cm horizontal bore magnet (GE NMR Instruments, Fremont, CA, USA) incorporating AVANCE digital electronics (Bruker Biospec, ParaVision 3.0.2 (OS); Bruker Medical, Billerica, MA, USA). Induction of anesthesia prior to imaging and maintenance of anesthesia during imaging was achieved by inhalation of isoflurane (~2-3% in oxygen). Anesthetized animals were placed on an acrylic sled for positioning within the magnet bore. Monitoring of the body temperature and physiological parameters during imaging was accomplished using an air heater system in conjunction with respiratory and temperature sensors located within the sled. Preliminary scout images were acquired on sagittal and coronal planes for slice positioning. Coronal T2-weighted images were acquired to enable visualization of orthotopic colon tumors. Data acquisition consisted of a localizer, T1-weighted MR images, and T2-weighted MR images. Anatomic coverage included the tumor, kidneys, and muscles. T1-weighted dynamic contrast-enhanced MRI (DCE-MRI) was performed using the intravascular contrast agent, albumin-gadopentetate dimeglumine (albumin-GdDTPA; University of California, San Francisco, CA, USA) (13). T1-relaxation rate measurements were performed using a saturation recovery, fast spin echo (FSE) sequence with an effective echo time of 10 ms, and a TR ranging from 360 to 6000 milliseconds [FOV=32×32 mm, slice thickness=1.0 mm, matrix size=128×96 pixels, number of excitations (NEX)=3. RARE factor=8]. Three coronal T1-FSE scans were acquired before contrast and five T1-FSE scans were acquired after administration of albumin-GdDTPA (0.1 mmol/kg). Image processing and analysis were carried out using commercially available software (ANALYZE PC, Version 7.0; Biomedical Imaging Resource, Mayo Foundation, Rochester, MN, USA) (Matlab's curve-fitting toolbox, Matlab Version 7.0; Math Works, Inc., Natick, MA, USA). Relative blood volume was calculated by linear regression analysis of the normalized change in T1-relaxation rate (ΔR1 tumor/blood ) of tumors as described by us previously (8, 13). Kidneys were sampled and used as a surrogate measure of the contrast agent concentration in the blood. ANTICANCER RESEARCH 31: 387-394 (2011) 388 Immunohistochemistry. CD31 immunostaining of untreated controls and MSC-treated tumors (0.2 mg/day × 14) was performed using previously described methods (8, 9). All CD31-positive intratumor microvessels were counted at ×400 magnification in each individual microscopic field on the viable parts of the entire tumor without any selection criteria. Single . Combined MRI-GFP FPI of GEO tumor response to selenium. The antitumor activity of selenium against orthotopic GEO tumors was examined by monitoring tumor growth over a 60-day period using combined MRI and FPI. (A) Coronal T2-weighted MR images of a control mouse and a selenium-treated mouse on day 60 post-implantation are shown on the left. Corresponding FPI (GFP) images of an animal from each group are also shown on the right. (B) Plot shows the change in volumes of control (n=6) and selenium-treated tumors (n=5) at different times postimplantation. Significant differences in tumor volume were seen on days 34 and 48 following implantation (p=0.01). eleven tumors (six controls, five MSC) in eight animals. DCE-MRI studies were performed on a total of sixteen tumors (eight controls, eight MSC) in eight animals. However, data from one MSC-treated animal (m5) were excluded from the analysis due to a poor contrastagent injection evidenced by lack of enhancement in the kidneys postcontrast. DCE-MRI data analysis was therefore performed using eight control tumors and six MSC-treated tumors. Immunohistochemical analysis was also performed on sixteen tumors (eight controls, eight MSC). Animals that did not reveal successful tumor 'take' were excluded from the study. A two-tailed Student's t-test was used for comparing treatment groups with untreated controls. P-values less than 0.05 were considered statistically significant. All statistical calculations and analyses were performed using Graph Pad Prism (Version 5.00; Graph Pad, San Diego, CA, USA). Results The progress of orthotopic colon tumors was visualized in nude mice using MRI and FPI. Serial FPI was performed once every three to four days beginning one week postimplantation. MRI was performed once a week. Whole-body FPI provided evidence of tumor growth seven days after surgical implantation even in the absence of a palpable tumor. As shown in In addition to anatomic imaging of tumor growth using MRI and FPI, the effects of selenium therapy on the vascularization of GEO colon tumors were assessed. To accomplish this, DCE-MRI was performed using an intravascular contrast agent, albumin-Gd-DTPA. DCE-MRI was performed on a total of eight animals (four control, four MSC) following daily treatment with selenium for a period of 17 days beginning day six after implantation. This time point was chosen based on the antiangiogenic efficacy of selenium observed in our previous studies using subcutaneous human tumor xenograft models To validate the findings obtained with MRI on the efficacy of selenium treatment on GEO vascular function, orthotopic GEO tumor-bearing mice were treated with MSC for a period of 14 days beginning 6 days after implantation. Tumors were excised and immunostained for CD31. Tumor sections obtained from control animals appeared to be well vascularized with an average MVD of 23.30±1.2 (n=8). Treatment with MSC led to a significant reduction in MVD (p<0.001; 9.50±0.5, n=8). Representative images of CD31-immunostained GEO tumor sections obtained from a control animal and an animal treated with 0.2 mg/kg MSC for 14 days are shown in The antitumor efficacy of selenium treatment was examined by monitoring tumor growth over a 60-day period using combined MRI and FPI. Coronal T2-weighted MR images of a control mouse and an MSC-treated mouse on day 60 are shown in Discussion Colorectal cancer is one of the leading causes of cancerrelated mortality in the United States (14). Given its biologic complexity and poor prognosis, it is important to evaluate novel treatment strategies that might potentially improve treatment outcome in patients. The essential dietary trace element, selenium, has been shown to act as an effective chemopreventive agent reducing the risk and mortality associated with cancer (15, 16). MSC is an organoselenium compound that has been shown to exert potent antiangiogenic activity in vivo Bhattacharya et al: Combination MRI and Fluorescence Imaging for Tumor Progression 391 Monitoring of subcutaneous (ectopic) tumors in small animals can be performed easily through the combination of visual inspection and simple caliper-based measurements of tumor dimension. In contrast, tumors established in orthotopic tumor sites cannot be assessed by visual examination and are often palpable only during advanced stages of tumor growth. It is widely recognized that orthotopic tumor models provide an appropriate simulation of the local tumor tissue microenvironment and are therefore better suited for preclinical evaluation of therapeutics Such multimodality imaging approaches have the potential to provide useful insight into the biology of orthotopic tumors and facilitate validation of tumor response to therapy. Therefore, in the present study, a dual-modality imaging approach was implemented using FPI and MRI to demonstrate the antiangiogenic and antitumor activity of selenium in an orthotopic model of human colon cancer in nude mice. Both FPI and MRI provided evidence of the therapeutic activity against the GEO colon adenocarcinomas. While whole-body FPI enabled high-throughput longitudinal monitoring of tumor growth and progression, MRI facilitated the quantification of vascular changes in the orthotopic colon tumors following therapeutic intervention. The complimentary nature of MRI and FPI allowed for simultaneous determination of tumor progression and angiogenesis and their response to therapy. MRI methods have been widely utilized in both preclinical and clinical settings for assessment of tumor response to therapy. Although FPI requires gene transfection of tumor cells which is currently not available clinically (21), fluorescence imaging techniques are being pursued for diagnostic clinical applications in oncology including early detection of neoplastic disease and mapping of sentinel lymph nodes with encouraging results In previous studies using subcutaneous tumors, the antiangiogenic efficacy of selenium has been observed consistently, particularly in well-vascularized, poorly differentiated tumors (9). Using immunohistochemistry, increased tumor vascular maturation following selenium treatment has also been demonstrated (8). The current study demonstrates the anti-angiogenic and antitumor activity of MSC in a clinically-relevant model. A significant decrease in MVD of orthotopic GEO colon tumors ( In the clinical setting, blocking VEGF alone (e.g. with bevacizumab) has met with limited therapeutic success in solid tumors, including colorectal cancer (24). In glioblastomas, an infiltrative phenotype with increased local invasion and distant metastasis has been observed after withdrawal of VEGF blockade In conclusion, the results of this study highlight the usefulness of multimodal imaging approaches in the assessment of tumor and angiogenic response to therapy and demonstrate the antiangiogenic effects of selenium in an orthotopic model of human colon cancer. However, a few limitations of the study need to be recognized. Although both MRI and FPI were used to monitor tumor progression and therapeutic response, only qualitative (visual) comparisons were made between the two imaging methods. However, previous studies have demonstrated good correlation between quantitative measurements of tumor volume and area obtained using MRI and FPI, respectively (6, Acknowledgements This research was supported by grants from the National Cancer Institute (NCI) 1R21 CA133682-01A2 (A.B.), Alliance Foundation (M.S) and utilized core resources supported by RPCI's Comprehensive Cancer Center Support Grant CA016056 from the NCI. Reference

Developing Folate-Conjugated miR-34a Therapeutic for Prostate Cancer: Challenges and Promises

Prostate cancer (PCa) remains a common cancer with high mortality in men due to its heterogeneity and the emergence of drug resistance. A critical factor contributing to its lethality is the presence of prostate cancer stem cells (PCSCs), which can self-renew, long-term propagate tumors, and mediate treatment resistance. MicroRNA-34a (miR-34a) has shown promise as an anti-PCSC therapeutic by targeting critical molecules involved in cancer stem cell (CSC) survival and functions. Despite extensive efforts, the development of miR-34a therapeutics still faces challenges, including non-specific delivery and delivery-associated toxicity. One emerging delivery approach is ligand-mediated conjugation, aiming to achieve specific delivery of miR-34a to cancer cells, thereby enhancing efficacy while minimizing toxicity. Folate-conjugated miR-34a (folate–miR-34a) has demonstrated promising anti-tumor efficacy in breast and lung cancers by targeting folate receptor α (FOLR1). Here, we first show that miR-34a, a TP53 transcriptional target, is reduced in PCa that harbors TP53 loss or mutations and that miR-34a mimic, when transfected into PCa cells, downregulated multiple miR-34a targets and inhibited cell growth. When exploring the therapeutic potential of folate–miR-34a, we found that folate–miR-34a exhibited impressive inhibitory effects on breast, ovarian, and cervical cancer cells but showed minimal effects on and targeted delivery to PCa cells due to a lack of appreciable expression of FOLR1 in PCa cells. Folate–miR-34a also did not display any apparent effect on PCa cells expressing prostate-specific membrane antigen (PMSA) despite the reported folate’s binding capability to PSMA. These results highlight challenges in the specific delivery of folate–miR-34a to PCa due to a lack of target (receptor) expression. Our study offers novel insights into the challenges and promises within the field and casts light on the development of ligand-conjugated miR-34a therapeutics for PCa

Effects of a novel peptide Ac-SDKP in radiation-induced coronary endothelial damage and resting myocardial blood flow

Abstract Background Cancer survivors treated with thoracic ionizing radiation are at higher risk of premature death due to myocardial ischemia. No therapy is currently available to prevent or mitigate these effects. We tested the hypothesis that an endogenous tetrapeptide N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP) counteracts radiation-induced coronary vascular fibrosis and endothelial cell loss and preserves myocardial blood flow. Methods We examined a rat model with external-beam-radiation exposure to the cardiac silhouette. We treated a subgroup of irradiated rats with subcutaneous Ac-SDKP for 18-weeks. We performed cardiac MRI with Gadolinium contrast to examine resting myocardial blood flow content. Upon sacrifice, we examined coronary endothelial-cell-density, fibrosis, apoptosis and endothelial tight-junction proteins (TJP). In vitro, we examined Ac-SDKP uptake by the endothelial cells and tested its effects on radiation-induced reactive oxygen species (ROS) generation. In vivo, we injected labeled Ac-SDKP intravenously and examined its endothelial localization after 4-h. Results We found that radiation exposure led to reduced resting myocardial blood flow content. There was concomitant endothelial cell loss and coronary fibrosis. Smaller vessels and capillaries showed more severe changes than larger vessels. Real-time PCR and confocal microscopy showed radiation-induced loss of TJ proteins including- claudin-1 and junctional adhesion molecule-2 (JAM-2). Ac-SDKP normalized myocardial blood flow content, inhibited endothelial cell loss, reduced coronary fibrosis and restored TJ-assembly. In vitro, Ac-SDKP localized to endothelial cells and inhibited radiation-induced endothelial ROS generation. In vivo, labeled Ac-SDKP was visualized into the endothelium 4-h after the intravenous injection. Conclusions We concluded that Ac-SDKP has protective effects against radiation-induced reduction of myocardial blood flow. Such protective effects are likely mediated by neutralization of ROS-mediated injury, preservation of endothelial integrity and inhibition of fibrosis. This demonstrates a strong therapeutic potential of Ac-SDKP to counteract radiotherapy-induced coronary disease

Phospholipid Encapsulation of an Anti-Fibrotic Endopeptide to Enhance Cellular Uptake and Myocardial Retention

Background: Cardioprotective effects of N-acetyl-ser-asp-lys-pro (Ac-SDKP) have been reported in preclinical models of myocardial remodeling. However, the rapid degradation of this endogenous peptide in vivo limits its clinical use. Method: To prolong its bioavailability, Ac-SDKP was encapsulated by phosphocholine lipid bilayers (liposomes) similar to mammalian cell membranes. The physical properties of the liposome structures were assessed by dynamic light scattering and scanning electron microscopy. The uptake of Ac-SDKP by RAW 264.7 macrophages and human and murine primary cardiac fibroblasts was confirmed by fluorescence microscopy and flow cytometry. Spectrum computerized tomography and competitive enzyme-linked immunoassays were performed to measure the ex vivo cardiac biodistribution of Ac-SDKP. The biological effects of this novel synthetic compound were examined in cultured macrophages and cardiac fibroblasts and in a murine model of acute myocardial infarction induced by permanent coronary artery ligation. Results: A liposome formulation resulted in the greater uptake of Ac-SDKP than the naked peptide by cultured RAW 264.7 macrophages and cardiac fibroblasts. Liposome-delivered Ac-SDKP decreased fibroinflammatory genes in cultured cardiac fibroblasts co-treated with TGF-β1 and macrophages stimulated with LPS. Serial tissue and serum immunoassays showed the high bioavailability of Ac-SDKP in mouse myocardium and in circulation. Liposome-delivered Ac-SDKP improved cardiac function and reduced myocardial fibroinflammatory responses in mice with acute myocardial infarction. Conclusion: Encapsulation of Ac-SDKP in a cell membrane-like phospholipid bilayer enhances its plasma and tissue bioavailability and offers cardioprotection against ischemic myocardial injury. Future clinical trials can use this novel approach to test small protective endogenous peptides in myocardial remodeling

Inactivation of hepatic enzymes by inhalant nitrite—In vivo and in vitro studies

We examined the effects of acute isobutyl nitrite (ISBN) exposure on the activity of several hepatic enzymes. Two strains of adult male mice (Balb/c and C57BL/6) were exposed to 900 ppm ISBN or ambient air for 45 minutes. The enzyme activity of hepatic cytochrome P450 (CYP)-mediated deethylation, glutathione S-transferase (GST), and carboxylesterase (CBE) was monitored through the substrates 3-cyano-7-ethoxycoumarin (CEC), 1-chloro-2,4-dinitrobenzene, and p-nitrophenyl acetate, respectively. Acute ISBN exposure led to a significant reduction in hepatic CYP-mediated CEC deethylation, GST, and CBE activity in Balb/c mice (of 81.5%, 74.7%, and 25.2%, respectively, vs control mice, each at P<.05) when livers were harvested immediately after inhalant exposure. The corresponding decreases in C57BL/6 mice were smaller (with reductions of 21.8%, 18.8%, and 13.3%, respectively, each at P<.05). This enzyme activity, tested in C57BL/6 mice only, returned to control values after a 24-hour period of nonexposure. Follow-up mechanistic investigations using rat liver GST indicated that ISBN-mediated enzyme inactivation was not caused by its metabolites: inorganic nitrite ion (NO2−) or nitric oxide. This inactivation could be prevented, but not reversed, by added glutathione, suggesting irreversible protein oxidation. Using different NO donors as comparative agents, we found that GST inactivation by ISBN was not associated with protein S-nitrosylation or disulfide formation, but with tyrosine nitration. Inhalant nitrite exposure, therefore, led to a significant reduction in hepatic enzyme activity in mice, possibly through tyrosine nitration of hepatic proteins. This effect raises the possibility of drug-drug metabolic interactions from inhalant nitrite abuse. However, determining the applicability of these findings to humans will require further study