J. Oliver McIntyre

4-Hydroxy-2(E)-nonenal inhibits CNS mitochondrial respiration at multiple sites

Abstract: A destructive cycle of oxidative stress and mitochondrial dysfunction is proposed in neurodegenerative disease. Lipid peroxidation, one outcome of oxidative challenge, can lead to the formation of 4‐hydroxy‐2(E)‐nonenal (HNE), a lipophilic alkenal that forms stable adducts on mitochondrial proteins. In this study, we characterized the effects of HNE on brain mitochondrial respiration. We used whole rat brain mitochondria and concentrations of HNE comparable to those measured in patients with Alzheimer’s disease. Our results showed that HNE inhibited respiration at multiple sites. Complex I‐linked and complex II‐linked state 3 respirations were inhibited by HNE with IC50 values of ∼200 μM HNE. Respiration was apparently diminished owing to the inhibition of complex III activity. In addition, complex II activity was reduced slightly. The lipophilicity and adduction characteristics of HNE were responsible for the effects of HNE on respiration. The inhibition of respiration was not prevented by N‐acetylcysteine or aminoguanidine. Studies using mitochondria isolated from porcine cerebral cortex also demonstrated an inhibition of complex I‐ and complex II‐linked respiration. Thus, in neurodegenerative disease, oxidative stress may impair mitochondrial respiration through the production of HNE.

Imaging matrix metalloproteinases in cancer.

Early detection of both primary tumors and metastatic disease remains a major challenge in the diagnosis and staging of cancer. The recognition of the role of MMPs in both the growth and metastasis of tumors has guided the development not only of therapeutic strategies utilizing synthetic, small-molecule MMP inhibitors (MMPIs), but has also catalyzed methods to detect and image tumors in vivo by means of tumor-associated proteolytic activity. These imaging approaches target MMPs involved in cancer progression via contrast agents linked to MMPIs or to MMP selective and specific substrates with sensitivity enhanced by amplification during enzymatic processing. This review draws attention to a variety of strategies utilized to image MMP activity in vivo.

Selective Visualization of Cyclooxygenase-2 in Inflammation and Cancer by Targeted Fluorescent Imaging Agents

Effective diagnosis of inflammation and cancer by molecular imaging is challenging because of interference from nonselective accumulation of the contrast agents in normal tissues. Here, we report a series of novel fluorescence imaging agents that efficiently target cyclooxygenase-2 (COX-2), which is normally absent from cells, but is found at high levels in inflammatory lesions and in many premalignant and malignant tumors. After either i.p. or i.v. injection, these reagents become highly enriched in inflamed or tumor tissue compared with normal tissue and this accumulation provides sufficient signal for in vivo fluorescence imaging. Further, we show that only the intact parent compound is found in the region of interest. COX-2-specific delivery was unambiguously confirmed using animals bearing targeted deletions of COX-2 and by blocking the COX-2 active site with high-affinity inhibitors in both in vitro and in vivo models. Because of their high specificity, contrast, and detectability, these fluorocoxibs are ideal candidates for detection of inflammatory lesions or early-stage COX-2-expressing human cancers, such as those in the esophagus, oropharynx, and colon.

Optical Imaging of Matrix Metalloproteinase-7 Activity In Vivo Using a Proteolytic Nanobeacon

Matrix metalloproteinases (MMPs) are extracellular proteolytic enzymes involved in tumor progression. We present the in vivo detection and quantitation of MMP7 activity using a specific near-infrared polymer-based proteolytic beacon, PB-M7NIR. PB-M7NIR is a pegylated polyamidoamine PAMAM-Generation 4 dendrimer core covalently coupled to a Cy5.5-labeled peptide representing a selective substrate that monitors MMP7 activity (sensor) and AF750 as an internal reference to monitor relative substrate concentration (reference). In vivo imaging of tumors expressing MMP7 had a median sensor to reference ratio 2.2-fold higher than a that of a bilateral control tumor. Ex vivo imaging of intestines of multiple intestinal neoplasia (APCMin) mice injected systemically with PB-M7NIR revealed a sixfold increase in the sensor to reference ratio in the adenomas of APCMin mice compared with control intestinal tissue or adenomas from MMP7-null Min mice. PB-M7NIR detected tumor sizes as small as 0.01 cm 2 , and the sensor to reference ratio was independent of tumor size. Histologic sectioning of xenograft tumors localized the proteolytic signal to the extracellular matrix; MMP7-overexpressing tumors displayed an approximately 300-fold enhancement in the sensor to reference ratio compared with nonexpressing tumor cells. In APCMin adenomas, the proteolytic signal colocalized with the endogenously expressed MMP7 protein, with sensor to reference ratios approximately sixfold greater than that of normal intestinal epithelium. PB-M7NIR provides a useful reagent for the in vivo and ex vivo quantitation and localization of MMP-selective proteolytic activity.

Increased diacylglycerols characterize hepatic lipid changes in progression of human nonalcoholic fatty liver disease; comparison to a murine model.

Background and Aims The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and progression to cirrhosis. While differences in liver lipids between disease states have been reported, precise composition of phospholipids and diacylglycerols (DAG) at a lipid species level has not been previously described. The goal of this study was to characterize changes in lipid species through progression of human NAFLD using advanced lipidomic technology and compare this with a murine model of early and advanced NAFLD. Methods Utilizing mass spectrometry lipidomics, over 250 phospholipid and diacylglycerol species (DAGs) were identified in normal and diseased human and murine liver extracts. Results Significant differences between phospholipid composition of normal and diseased livers were demonstrated, notably among DAG species, consistent with previous reports that DAG transferases are involved in the progression of NAFLD and liver fibrosis. In addition, a novel phospholipid species (ether linked phosphatidylinositol) was identified in human cirrhotic liver extracts. Conclusions Using parallel lipidomics analysis of murine and human liver tissues it was determined that mice maintained on a high-fat diet provide a reproducible model of NAFLD in regards to specificity of lipid species in the liver. These studies demonstrated that novel lipid species may serve as markers of advanced liver disease and importantly, marked increases in DAG species are a hallmark of NAFLD. Elevated DAGs may contribute to altered triglyceride, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) levels characteristic of the disease and specific DAG species might be important lipid signaling molecules in the progression of NAFLD.

Molecular imaging of proteolytic activity in cancer

The early detection of both primary tumors and metastatic disease continue to be significant challenges in the diagnosis and staging of cancer. The growing recognition of the role of proteinases and proteolytic cascades in both the growth and metastasis of tumors has led to the development not only of therapeutic strategies using proteinase inhibitors, but also of methods to detect and image tumors in vivo via tumor‐associated proteolytic activities. These imaging strategies derive from the enhanced sensitivity afforded by amplification that can be obtained by enzymatic processing to increase the efficacy of imaging “contrast agents” coupled with the inherent substrate specificity and selectivity of proteinases. This review describes key proteinases important in cancer progression, the strategies that have been devised to detect and image proteolytic activity in vivo, and the potential for this kind of functional imaging to serve as a marker for targeted therapy. The intent is to draw attention to the developing methods of molecular imaging to facilitate not only cancer diagnosis, but also for devising strategies for individualized targeted therapy and non‐invasive monitoring of therapeutic efficacy.

[3] Large-scale preparation of rat liver mitochondria in high yield

Publisher Summary Preparation of mitochondrial components requires large quantities of mitochondria. This chapter describes a procedure for large-scale isolation of rat liver mitochondria in high yield. Mitochondria are recovered from the nuclear fraction and the postnuclear supernatant. The chapter discusses the characterization and yield of the mitochondria. The recovery of mitochondria (both mitochondria R1 and R2) is approximately two-thirds of the amount estimated to be in the liver. Mitochondria R1 are intact and are mainly in the condensed configuration. They approach ultimate purity, as judged by electron microscopy and analysis using marker enzymes. They are practically devoid of contamination by other organelles, such as endoplasmic reticulum, plasma membranes, peroxisomes, and lysosomes, which contaminate mitochondria R2. Mitochondria R2 are estimated to be 80–85% pure. The major contamination, about 10–15%, is by endoplasmic reticulum.

The orientation of d-β-hydroxybutyrate dehydrogenase in the mitochondrial inner membrane

Abstract d -β-Hydroxybutyrate dehydrogenase of beef heart mitochondria is a lipid-requiring enzyme, bound to the inner membrane. The orientation of this enzyme in the membrane has been studied by comparing the characteristics of the enzyme in mitochondria and ‘inside-out’ submitochondrial vesicles. We observe that the enzymic activity is (1) latent in intact mitochondria; (2) relatively stable to trypsin digestion in mitochondria but rapidly inactivated in submitochondrial vesicles by this treatment; and (3) released more rapidly from submitochondrial vesicles by phospholipase A2 digestion than from mitochondria. Conclusive evidence that d -β-hydroxybutyrate dehydrogenase is localized on the matrix face of the mitochondrial inner membrane is provided by the correlation that the enzyme is released from submitochondrial vesicles before the membrane becomes leaky to cytochrome c . The arrangement of d -β-hydroxybutyrate dehydrogenase in the membrane is discussed within a generalized classification of the orientation of proteins in membranes. The evidence indicates that d -β-hydroxybutyrate dehydrogenase is an amphipathic molecule and as such is inlaid in the membrane, i.e. the enzyme is partially inserted into the hydrophobic milieu of the membrane, with the polar, functional end extending into the aqueous milieu.

Novel solubility-switchable MRI agent allows the noninvasive detection of matrix metalloproteinase-2 activity in vivo in a mouse model

A novel MRI proteinase‐modulated contrast agent (PCA) was developed to detect the activity of the proinvasive enzyme matrix metalloproteinase‐2 (MMP‐2) in vivo. The PCA2‐switch agent incorporates a solubility switch, where cleavage of a peptide substrate by MMP‐2 decreases the water solubility of the agent. Evidence suggests that this leads to an accumulation of cleaved PCA2‐switch in an MMP‐2‐positive, wild‐type, MC7‐L1 mammary carcinoma tumor in a Balb/c mouse model compared to a MC7‐L1 MMP‐2‐knockdown tumor. When a scrambled peptide sequence is inserted into the agent (PCA2‐scrambled), the in vitro cleavage efficiency of MMP‐2 is markedly reduced. In vivo, PCA2‐scrambled does not accumulate in the wild‐type tumor and the pharmacokinetics is similar in both tumors. In conclusion, in vivo cleavage of PCA2‐switch by MMP‐2 results in a significant accumulation of the cleaved PCA2‐switch in an MMP‐2‐positive tumor. Magn Reson Med 60:1056–1065, 2008.

Noninvasive Detection of Matrix Metalloproteinase Activity In Vivo using a Novel Magnetic Resonance Imaging Contrast Agent with a Solubility Switch

We have developed novel proteinase-modulated contrast agents (PCAs) to detect the activity of proteinases in vivo using magnetic resonance imaging. The PCAs are based on the concept of a solubility switch, from hydrophilic to hydrophobic, that significantly modifies the pharmacokinetic properties of the agent as revealed by the slow efflux kinetics from the activity site. Our compound PCA7-switch detects the activity of the secreted matrix-degrading proteinase matrix-metalloproteinase 7 (MMP-7) in living, tumor-bearing mice. Control experiments were performed using an agent that was not cleaved by MMP-7 (PCA7-scrambled), an agent that could be cleaved by MMP-7 but lacked the solubility switch (PCA7-B), and a standard contrast agent (gadolinium–diethylenetriaminepentaacetic acid). PCA7-switch detected a reduction in MMP-7 activity in tumor-bearing mice treated with a synthetic MMP inhibitor, demonstrating its effectiveness in noninvasive functional imaging of proteolytic activity in vivo.