Alkaline surface treatment and time-resolved reading of mn-doped nanocrystal signal transducer for enhanced bioassay sensitivity.

Recently, Mn-doped semiconductor nanocrystals (NCs) with high brightness, long lifetimes, and low-energy excitation are emerging for time-resolved luminescence biosensing/imaging. Following our previous work on Mn-doped NCs, in this work we developed poly(styrene-co-maleic anhydride) (PSMA)-encapsulated Mn-doped AgZnInS/ZnS NCs as signal transducers for immunoassay of capsular polysaccharide (CPS), a surface antigen and also a biomarker of Burkholderia pseudomallei which causes a fatal disease called melioidosis. To enhance the assay sensitivity, a surface treatment for PSMA-encapsulated NCs (NC-probes) was performed to promote the presence of carboxyl groups that help conjugate more anti-CPS antibodies to the surface of NC-probes and thus enhance bioassay signals. Meanwhile, time-resolved reading on the luminescence of NC-probes was adopted to minimize the assay background autofluorescence. Both strategies essentially enhance the assay signal-to-background ratio (or equivalently the assay sensitivity) by increasing the signal and decreasing the background, respectively. Through performing and comparing immunoassays with different NC-probes (with and without surface treatment) and different signal reading methods (time-resolved reading and non-time-resolved reading), it was proven that the immunoassay adopting surface-treated NC-probes and time-resolved reading achieved a lower limit-of-detection (LOD) than the ones adopting non-surface-treated NC-probes or non-time-resolved reading. Moreover, the achieved LOD is comparable to the LOD of immunoassay using enzyme horseradish peroxidase as a signal transducer.

3-Methylglutarylcarnitine: A biomarker of mitochondrial dysfunction.

The acylcarnitines comprise a wide range of acyl groups linked via an ester bond to the hydroxyl group of L-carnitine. Mass spectrometry methods are capable of measuring the relative abundance of hundreds of acylcarnitines in a single drop of blood. As such, acylcarnitines can serve as sensitive biomarkers of disease. For certain acylcarnitines, however, their biochemical origin, and biomedical significance, remain unclear. One such example is 3-methylglutaryl (3MG) carnitine (C5-3M-DC). Whereas 3MG carnitine levels are normally very low, elevated levels are detected in discrete inborn errors of metabolism (IEM) as well as different forms of heart disease. Moreover, acute injury, including γ radiation exposure, paraquat poisoning, and traumatic brain injury manifest elevated levels of 3MG carnitine in blood and/or urine. Recent evidence indicates that two distinct biosynthetic routes to 3MG carnitine exist. The first, caused by an inherited deficiency in the leucine catabolism pathway enzyme, 3-hydroxy-3-methylglutaryl (HMG) CoA lyase, leads to a buildup of trans-3-methylglutaconyl (3MGC) CoA. Reduction of the double bond in trans-3MGC CoA generates 3MG CoA, which is then converted to 3MG carnitine by carnitine acyltransferase. This route, however, cannot explain why 3MG carnitine levels increase in IEMs that do not affect leucine metabolism or various chronic and acute disease states. In these cases, disease-related defects in aerobic energy metabolism result in diversion of acetyl CoA to trans-3MGC CoA. Once formed, trans-3MGC CoA is reduced to 3MG CoA and esterified to form 3MG carnitine. Thus, 3MG carnitine, represents a potential biomarker of disease processes associated with compromised mitochondrial energy metabolism.

Characterization of trans-3-Methylglutaconyl CoA-Dependent Protein Acylation.

3-methylglutaconyl (3MGC) CoA hydratase (AUH) is the leucine catabolism pathway enzyme that catalyzes the hydration of trans-3MGC CoA to 3-hydroxy, 3-methylglutaryl (HMG) CoA. In several inborn errors of metabolism (IEM), however, metabolic dysfunction can drive this reaction in the opposite direction (the dehydration of HMG CoA). The recent discovery that trans-3MGC CoA is inherently unstable and prone to a series of non-enzymatic chemical reactions provides an explanation for 3MGC aciduria observed in these IEMs. Under physiological conditions, trans-3MGC CoA can isomerize to cis-3MGC CoA, which is structurally poised to undergo intramolecular cyclization with the loss of CoA, generating cis-3MGC anhydride. The anhydride is reactive and has two potential fates; (a) hydrolysis to yield cis-3MGC acid or (b) a reaction with lysine side-chain amino groups to 3MGCylate substrate proteins. An antibody elicited against a 3MGC hapten was employed to investigate protein acylation in incubations containing recombinant AUH, HMG CoA, and bovine serum albumin (BSA). The data obtained show that, as AUH dehydrates HMG CoA to trans-3MGC CoA, BSA is acylated. Moreover, α-3MGC IgG immunoblot signal intensity correlates with AUH concentration, HMG CoA substrate concentration, and incubation time. Thus, protein 3MGCylation may contribute to the phenotypic features associated with IEMs that manifest 3MGC aciduria.

Isolation of recombinant apolipoprotein E4 N-terminal domain by foam fractionation.

Apolipoprotein (apo) E functions in lipoprotein metabolism as a low density lipoprotein receptor ligand. ApoE is comprised of two structural domains, a 22 kDa N-terminal (NT) domain that adopts a helix bundle conformation and a 10 kDa C-terminal domain with strong lipid binding affinity. The NT domain is capable of transforming aqueous phospholipid dispersions into discoidal reconstituted high density lipoprotein (rHDL) particles. Given the utility of apoE-NT as a structural component of rHDL, expression studies were conducted. A plasmid construct encoding a pelB leader sequence fused to the N-terminus of human apoE4 (residues 1-183) was transformed into Escherichia coli. Upon expression, the fusion protein is directed to the periplasmic space where leader peptidase cleaves the pelB sequence, generating mature apoE4-NT. In shaker flask expression cultures, apoE4-NT escapes the bacteria and accumulates in the medium. In a bioreactor setting, however, apoE4-NT was found to combine with gas and liquid components in the culture medium to generate large quantities of foam. When this foam was collected in an external vessel and collapsed into a liquid foamate, analysis revealed that apoE4-NT was the sole major protein present. The product protein was further isolated by heparin affinity chromatography (60-80 mg/liter bacterial culture), shown to be active in rHDL formulation, and documented to serve as an acceptor of effluxed cellular cholesterol. Thus, foam fractionation provides a streamlined process to produce recombinant apoE4-NT for biotechnology applications.

Formulation and Characterization of Bioactive Agent Containing Nanodisks.

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle's lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin B as the hydrophobic bioactive agent.

Cardiac-specific deficiency of 3-hydroxy-3-methylglutaryl coenzyme A lyase in mice causes cardiomyopathy and a distinct pattern of acyl-coenzyme A-related biomarkers.

Deficiency of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase (HL) is an autosomal recessive inborn error of acyl-CoA metabolism affecting the last step of leucine degradation. Patients with HL deficiency (HLD) can develop a potentially fatal cardiomyopathy. We created mice with cardiomyocyte-specific HLD (HLHKO mice), inducing Cre recombinase-mediated deletion of exon 2 at two months of age. HLHKO mice survive, but develop left ventricular hypertrophy by 9 months. Also, within minutes after intraperitoneal injection of the leucine metabolite 2-ketoisocaproate (KIC), they show transient left ventricular hypocontractility and dilation. Leucine-related acyl-CoAs were elevated in HLHKO heart (e.g., HMG-CoA, 34.0 ± 4.4 nmol/g versus 0.211 ± 0.041 in controls, p < 0.001; 3-methylcrotonyl-CoA, 5.84 ± 0.69 nmol/g versus 0.282 ± 0.043, p < 0.001; isovaleryl-CoA, 1.86 ± 0.30 nmol/g versus 0.024 ± 0.014, p < 0.01), a similar pattern to that in liver of mice with hepatic HL deficiency. After KIC loading, HMG-CoA levels in HLHKO heart were higher than under basal conditions, as were the ratios of HMG-CoA/acetyl-CoA and of HMG-CoA/succinyl-CoA. In contrast to the high levels of multiple leucine-related acyl-CoAs, biomarkers in urine and plasma of HLHKO mice show isolated hyper-3-methylglutaconic aciduria (700.8 ± 48.4 mmol/mol creatinine versus 37.6 ± 2.4 in controls, p < 0.001), and elevated C5-hydroxyacylcarnitine in plasma (0.248 ± 0.014 µmol/L versus 0.048 ± 0.005 in controls, p < 0.001). Mice with liver-specific HLD were compared, and showed normal echocardiographic findings and normal acyl-CoA profiles in heart. This study of nonhepatic tissue-specific HLD outside of liver reveals organ-specific origins of diagnostic biomarkers for HLD in blood and urine and shows that mouse cardiac HL is essential for myocardial function in a cell-autonomous, organ-autonomous fashion.

Studies of the cardiolipin interactome.

Cardiolipin (CL) is a unique phospholipid that is fundamental to the structure and function of the highly curved cristae membranes of mitochondria. Given its distinctive cone-shaped molecular architecture, CL induces negative membrane curvature in a bilayer setting. Another key feature of CL is its intrinsic ability to interact with various ligands, including cytochrome c, the anti-neoplastic anthracycline, doxorubicin, and the divalent cation, calcium. Although these, and other, binding interactions exert profound effects on mitochondrial and cellular function, they are difficult to study in intact mitochondria. Whereas liposomes provide a potential model membrane system, their relatively large size, limited ability to accommodate CL and the presence of an inaccessible interior bilayer leaflet, make these structures suboptimal. The discovery that CL can be formulated into aqueous soluble, reconstituted high density lipoprotein particles, termed nanodisks (ND), provides an alternative model membrane system. Comprised solely of CL and an apolipoprotein scaffold, CL-ND exist as a disk-shaped phospholipid bilayer whose perimeter is stabilized by contact with the scaffold protein. In these nanoscale particles, both leaflets of the bilayer are solvent accessible, an advantage for studies of ligand interactions. Recent experiments employing CL-ND have yielded novel insight into apoptosis, cardiotoxicity and CL-dependent bilayer to non-bilayer transitions.

Cardiolipin nanodisks confer protection against doxorubicin-induced mitochondrial dysfunction.

Doxorubicin (DOX) is an aqueous soluble anthracycline therapeutic widely used in cancer treatment. Although DOX anti-cancer activity is dose-dependent, increased dosage enhances the risk of cardiotoxicity. Despite intensive investigation, the molecular basis of this undesirable side effect has yet to be established. In addition to serving as a DNA intercalation agent, DOX is known to bind to the signature mitochondrial phospholipid, cardiolipin (CL). Consistent with this, DOX associates with aqueous soluble nanoparticles, termed nanodisks (ND), comprised solely of CL and an apolipoprotein scaffold. Fluorescence microscopy analysis revealed that DOX uptake, and targeting to the nucleus of cultured hepatocarcinoma (HepG2) or breast cancer (MCF7) cells, was unaffected by its association with CL-ND. Subsequent studies revealed that free DOX and DOX-CL-ND were equivalent in terms of growth inhibition activity in both cell lines. By contrast, in studies with H9C2 cardiomyocytes, DOX-CL-ND induced a lesser concentration-dependent decline in cell viability than free DOX. Whereas incubation of H9C2 cardiomyocytes with free DOX caused a steep decline in maximal oxygen consumption rate, DOX-CL-ND treated cells were largely unaffected. The data indicate that association of DOX with CL-ND does not diminish its cancer cell growth inhibition activity yet confers protection to cardiomyocytes from DOX-induced effects on aerobic respiration. This study illustrates that interaction with CL plays a role in DOX-induced mitochondrial dysfunction and suggests CL-ND provide a tool for investigating the mechanistic basis of DOX-induced cardiotoxicity.

Diversion of Acetyl CoA to 3-Methylglutaconic Acid Caused by Discrete Inborn Errors of Metabolism.

A growing number of inborn errors of metabolism (IEM) have been identified that manifest 3-methylglutaconic (3MGC) aciduria as a phenotypic feature. In primary 3MGC aciduria, IEM-dependent deficiencies in leucine pathway enzymes prevent catabolism of trans-3MGC CoA. Consequently, this metabolite is converted to 3MGC acid and excreted in urine. In secondary 3MGC aciduria, however, no leucine metabolism pathway enzyme deficiencies exist. These IEMs affect mitochondrial membrane structure, electron transport chain function or ATP synthase subunits. As a result, acetyl CoA oxidation via the TCA cycle slows and acetyl CoA is diverted to trans-3MGC CoA, and then to 3MGC acid. Whereas the trans diastereomer of 3MGC CoA is the only biologically relevant diastereomer, the urine of affected subjects contains both cis- and trans-3MGC acids. Studies have revealed that trans-3MGC CoA is susceptible to isomerization to cis-3MGC CoA. Once formed, cis-3MGC CoA undergoes intramolecular cyclization, forming an anhydride that, upon hydrolysis, yields cis-3MGC acid. Alternatively, cis-3MGC anhydride can acylate protein lysine side chains. Once formed, cis-3MGCylated proteins can be deacylated by the NAD+-dependent enzyme, sirtuin 4. Taken together, the excretion of 3MGC acid in secondary 3MGC aciduria represents a barometer of defective mitochondrial function.

Foam fractionation of a recombinant biosurfactant apolipoprotein.

Locusta migratoria apolipophorin III (apoLp-III) possesses the ability to exist as a water soluble amphipathic α-helix bundle and a lipid surface seeking apolipoprotein. The intrinsic ability of apoLp-III to transform phospholipid vesicles into reconstituted discoidal high-density lipoproteins (rHDL) has led to myriad applications. To improve the yield of recombinant apoLp-III, studies were performed in a bioreactor. Induction of apoLp-III expression generated a protein product that is secreted from E. coli into the culture medium. Interaction of apoLp-III with gas and liquid components in media produced large quantities of thick foam. A continuous foam fractionation process yielded a foamate containing apoLp-III as the sole major protein component. The yield of recombinant apoLp-III was ~0.2 g / liter bacterial culture. Mass spectrometry analysis verified the identity of the target protein and indicated no modifications or changes to apoLp-III occurred as a result of foam fractionation. The functional ability of apoLp-III to induce rHDL formation was evaluated by incubating foam fractionated apoLp-III with phosphatidylcholine vesicles. FPLC size exclusion chromatography revealed a single major population of particles in the size range of rHDL. The results described offer a novel approach to bioreactor-based apoLp-III production that takes advantage of its intrinsic biosurfactant properties.

Role of non-enzymatic chemical reactions in 3-methylglutaconic aciduria.

3-Methylglutaconic (3MGC) aciduria occurs in numerous inborn errors associated with compromised mitochondrial energy metabolism. In these disorders, 3MGC CoA is produced de novo from acetyl CoA in three steps with the final reaction catalysed by 3MGC CoA hydratase (AUH). In in vitro assays, whereas recombinant AUH dehydrated 3-hydroxy-3-methylglutaryl (HMG) CoA to 3MGC CoA, free CoA was also produced. Although HMG CoA is known to undergo non-enzymatic intramolecular cyclisation, forming HMG anhydride and free CoA, the amount of free CoA generated increased when AUH was present. To test the hypothesis that the AUH-dependent increase in CoA production is caused by intramolecular cyclisation of 3MGC CoA, gas chromatography-mass spectrometry analysis of organic acids was performed. In the absence of AUH, HMG CoA was converted to HMG acid while, in the presence of AUH, 3MGC acid was also detected. To determine which 3MGC acid diastereomer was formed, immunoblot assays were conducted with 3MGCylated BSA. In competition experiments, when α-3MGC IgG was preincubated with trans-3MGC acid or cis-3MGC acid, the cis diastereomer inhibited antibody binding to 3MGCylated BSA. When an AUH assay product mix served as competitor, α-3MGC IgG binding to 3MGCylated BSA was also inhibited, indicating cis-3MGC acid is produced in incubations of AUH and HMG CoA. Thus, non-enzymatic isomerisation of trans-3MGC CoA drives AUH-dependent HMG CoA dehydration and explains the occurrence of cis-3MGC acid in urine of subjects with 3MGC aciduria. Furthermore, the ability of cis-3MGC anhydride to non-enzymatically acylate protein substrates may have deleterious pathophysiological consequences.

Lutein nanodisks protect human retinal pigment epithelial cells from UV light-induced damage.

The hydrophobic carotenoid, lutein, was conferred with aqueous solubility upon formulation into reconstituted discoidal high density lipoprotein particles, termed lutein nanodisks (ND). When formulated with phosphatidylcholine (PC), apolipoprotein (apo) A-I and lutein (formulation ratio = 5 mg PC/2 mg apoA-I/1 mg lutein), lutein solubilization efficiency in phosphate buffered saline (PBS) was ~90%. The UV/Vis absorbance maxima for lutein ND in PBS were red shifted by 6-13 nm versus the corresponding lutein absorbance maxima in ethanol. FPLC gel filtration chromatography gave rise to a single major absorbance peak in the size range of ND. Incubation of cultured ARPE-19 cells with lutein ND resulted in lutein uptake, as determined by HPLC analysis of cell extracts. Compared to control incubations, ARPE-19 cells incubated with lutein ND were protected from UV light-induced loss of cell viability. Consistent with this, reactive oxygen species generation, induced by exposure to UV irradiation, was lower in lutein-enriched cells than in control cells. Thus, uptake of ND-associated lutein protects ARPE-19 cells from UV light-induced damage. Taken together, the data indicate ND provide an aqueous lutein delivery vehicle for biotechnological or therapeutic applications.

Calcium-induced release of cytochrome c from cardiolipin nanodisks: Implications for apoptosis.

Miniature bilayer membranes comprised of phospholipid and an apolipoprotein scaffold, termed nanodisks (ND), have been used in binding studies. When ND formulated with cardiolipin (CL), but not phosphatidylcholine, were incubated with cytochrome c, FPLC gel filtration chromatography provided evidence of a stable binding interaction. Incubation of CL ND with CaCl2 resulted in a concentration-dependent increase in sample turbidity caused by ND particle disruption. Prior incubation of CL ND with cytochrome c increased CL ND sensitivity to CaCl2-induced effects. Centrifugation of CaCl2-treated CL ND samples yielded pellet and supernatant fractions. Whereas the ND scaffold protein, apolipophorin III, was recovered in the pellet fraction along with CL, the majority of the cytochrome c pool was in the supernatant fraction. Moreover, when cytochrome c CL ND were incubated with CaCl2 at concentrations below the threshold to induce ND particle disruption, FPLC analysis showed that cytochrome c was released. Pre-incubation of CL ND with CaCl2 under conditions that do not disrupt ND particle integrity prevented cytochrome c binding to CL ND. Thus, competition between Ca2+ and cytochrome c for a common binding site on CL modulates cytochrome c binding and likely plays a role in its dissociation from CL-rich cristae membranes in response to apoptotic stimuli.

Reconstituted HDL as a therapeutic delivery device.

Studies of "pre ß" high density lipoprotein (HDL) and reconstituted HDL (rHDL) have contributed to our understanding of the Reverse Cholesterol Transport pathway. The relative ease with which discoidal rHDL can be generated in vitro has led to novel applications including a) infusion of rHDL into patients to promote regression of atherosclerosis; b) use of rHDL as a miniature membrane for integration of transmembrane proteins in a native-like conformation and c) incorporation of hydrophobic bioactive molecules into rHDL, creating a delivery device. The present review is focused on bioactive agent containing rHDL. The broad array of hydrophobic bioactive molecules successfully incorporated into these particles is discussed, as well as the use of natural lipids and synthetic lipid analogs to confer distinctive binding activity. This technology remains in its infancy with the full potential of these simple, yet elegant, nanoparticles still to be discovered.

Inborn errors of metabolism associated with 3-methylglutaconic aciduria.

A growing number of inborn errors of metabolism (IEM) associated with compromised mitochondrial energy metabolism manifest an unusual phenotypic feature: 3-methylglutaconic (3MGC) aciduria. Two major categories of 3MGC aciduria, primary and secondary, have been described. In primary 3MGC aciduria, IEMs in 3MGC CoA hydratase (AUH) or HMG CoA lyase block leucine catabolism, resulting in a buildup of pathway intermediates, including 3MGC CoA. Subsequent thioester hydrolysis yields 3MGC acid, which is excreted in urine. In secondary 3MGC aciduria, no deficiencies in leucine catabolism enzymes exist and 3MGC CoA is formed de novo from acetyl CoA. In the "acetyl CoA diversion pathway", when IEMs directly, or indirectly, interfere with TCA cycle activity, acetyl CoA accumulates in the matrix space. This leads to condensation of two acetyl CoA to form acetoacetyl CoA, followed by another condensation between acetyl CoA and acetoacetyl CoA to form 3-hydroxy, 3-methylglutaryl (HMG) CoA. Once formed, HMG CoA serves as a substrate for AUH, producing trans-3MGC CoA. Non enzymatic isomerization of trans-3MGC CoA to cis-3MGC CoA precedes intramolecular cyclization to cis-3MGC anhydride plus CoA. Subsequent hydrolysis of cis-3MGC anhydride gives rise to cis-3MGC acid, which is excreted in urine. In reviewing 20 discrete IEMs that manifest secondary 3MGC aciduria, evidence supporting the acetyl CoA diversion pathway was obtained. This biochemical pathway serves as an "overflow valve" in muscle / brain tissue to redirect acetyl CoA to 3MGC CoA when entry to the TCA cycle is impeded.

Coenzyme Q nanodisks counteract the effect of statins on C2C12 myotubes.

Depletion of coenzyme Q (CoQ) is associated with disease, ranging from myopathy to heart failure. To induce a CoQ deficit, C2C12 myotubes were incubated with high dose simvastatin. This resulted in a concentration-dependent inhibition of cell viability. Simvastatin-induced effects were prevented by co-incubation with mevalonic acid. When myotubes were incubated with 60 µM simvastatin, mitochondrial CoQ content decreased while co-incubation with CoQ nanodisks (ND) increased mitochondrial CoQ levels and improved cell viability. Incubation of myotubes with simvastatin also led to a reduction in oxygen consumption rate (OCR). When myotubes were co-incubated with simvastatin and CoQ ND, the decline in OCR was ameliorated. The data indicate that CoQ ND represent a water soluble vehicle capable of delivering CoQ to cultured myotubes. Thus, these biocompatible nanoparticles have the potential to bypass poor CoQ oral bioavailability as a treatment option for individuals with severe CoQ deficiency syndromes and/or aging-related CoQ depletion.

Isomerization of trans-3-methylglutaconic acid.

3-Methylglutaconic (3MGC) aciduria is a common phenotypic feature of a growing number of inborn errors of metabolism. "Primary" 3MGC aciduria is caused by deficiencies in leucine pathway enzymes while "secondary" 3MGC aciduria results from inborn errors of metabolism that impact mitochondrial energy production. The metabolic precursor of 3MGC acid is trans-3MGC CoA, an intermediate in the leucine catabolism pathway. Gas chromatography-mass spectrometry (GC-MS) analysis of commercially available trans-3MGC acid yielded a mixture of cis and trans isomers while 1H-NMR spectroscopy of trans-3MGC acid at 25°C provided no evidence for the cis isomer. When trans-3MGC acid was incubated under conditions used for sample derivatization prior to GC-MS (but with no trimethylsilane added), 1H-NMR spectroscopy provided evidence of trans to cis isomerization. Incubation of trans-3MGC acid at 37°C resulted in time-dependent isomerization to cis-3MGC acid. Cis-3MGC acid behaved in a similar manner except that, under identical incubation conditions, less isomerization occurred. In agreement with these experimental results, molecular modeling studies provided evidence that the energy minimized structure of cis-3MGC acid is 4 kJ/mol more stable than that for trans-3MGC acid. Once generated in vivo, trans-3MGC acid is proposed to isomerize via a mechanism involving π electron delocalization with formation of a resonance structure that permits bond rotation. The data presented are consistent with the occurrence of both diastereomers in urine samples of subjects with 3MGC aciduria.

trans-3-Methylglutaconyl CoA isomerization-dependent protein acylation.

3-methylglutaconic (3MGC) aciduria is associated with a growing number of discrete inborn errors of metabolism. Herein, an antibody-based approach to detection/quantitation of 3MGC acid has been pursued. When trans-3MGC acid conjugated keyhole limpet hemocyanin (KLH) was inoculated into rabbits a strong immune response was elicited. Western blot analysis provided evidence that immune serum, but not pre-immune serum, recognized 3MGC-conjugated bovine serum albumin (BSA). In competition ELISAs using isolated immune IgG, the limit of detection for free trans-3MGC acid was compared to that for cis-3MGC acid and four structurally related short-chain dicarboxylic acids. Surprisingly, cis-3MGC acid yielded a much lower limit of detection (∼0.1 mg/ml) than trans-3MGC acid (∼1.0 mg/ml) while all other dicarboxylic acids tested were poor competitors. The data suggest trans-3MGC- isomerized during, or after, conjugation to KLH such that the immunogen was actually comprised of KLH harboring a mixture of cis- and trans-3MGC haptens. To investigate this unexpected isomerization reaction, trans-3MGC CoA was prepared and incubated at 37 °C in the presence of BSA. Evidence was obtained that non-enzymatic isomerization of trans-3MGC CoA to cis-3MGC CoA precedes intramolecular catalysis to form cis-3MGC anhydride plus CoASH. Anhydride-dependent acylation of BSA generated 3MGCylated BSA, as detected by anti-3MGC immunoblot. The results presented provide an explanation for the unanticipated detection of 3MGCylated proteins in a murine model of primary 3MGC aciduria. Furthermore, non-enzymatic hydrolysis of cis-3MGC anhydride represents a potential source of cis-3MGC acid found in urine of subjects with 3MGC aciduria.

Dye binding assay reveals doxorubicin preference for DNA versus cardiolipin.

Doxorubicin (DOX) is a potent anticancer agent that binds both DNA and cardiolipin (CL). To investigate DOX binding to CL versus DNA, aqueous soluble, CL-enriched nanoparticles, termed nanodisks (ND), were employed. Upon incubation with CL-ND, but not with phosphatidylcholine ND, DOX binding was detected. DOX binding to CL-ND was sensitive to buffer pH and ionic strength. To investigate if a DOX binding preference for DNA versus CL-ND exists, an agarose gel-based dye binding assay was developed. Under conditions wherein the commercial fluorescent dye, GelRed, detects a 636 bp DNA template following electrophoresis, DOX staining failed to visualize this DNA band. Incubation of the template DNA with DOX prior to electrophoresis resulted in a DOX concentration-dependent attenuation of GelRed staining intensity. When the template DNA was pre-incubated with equivalent amounts of free DOX or DOX-CL-ND, no differences in the extent of GelRed staining intensity attenuation were noted. When DOX was incubated with DNA alone, or a mixture of DNA and CL-ND, the extent of DOX-induced GelRed staining intensity attenuation was equivalent. Thus, DOX has a binding preference for DNA versus CL and, moreover, DOX-CL-ND offer a potential strategy to prevent DOX-induced cardiotoxicity while not affecting its affinity for DNA.

Assembly and Characterization of Biocompatible Coenzyme Q10 -Enriched Lipid Nanoparticles.

Coenzyme Q10 (CoQ10 ) is a strongly hydrophobic lipid that functions in the electron transport chain and as an antioxidant. CoQ10 was conferred with aqueous solubility by incorporation into nanoparticles containing phosphatidylcholine (PtdCho) and apolipoprotein (apo) A-I. These particles, termed CoQ10 nanodisks (ND), contain 1.0 mg CoQ10 /5 mg PtdCho/2 mg apoA-I (97% CoQ10 solubilization efficiency). UV/Vis absorbance spectroscopy of CoQ10 ND revealed a characteristic absorbance peak centered at 275 nm. Incorporation of CoQ10 into ND resulted in quenching of apoA-I tryptophan fluorescence emission. Gel filtration chromatography of CoQ10 ND gave rise to a single major absorbance peak and HPLC of material extracted from this peak confirmed the presence of CoQ10 . Incubation of cultured cells with CoQ10 ND, but not empty ND, resulted in a significant increase in the CoQ10 content of mitochondria as well as enhanced oxidative phosphorylation, as observed by a ~24% increase in maximal oxygen consumption rate. Collectively, a facile method to solubilize significant quantities of CoQ10 in lipid nanoparticles has been developed. The availability of CoQ10 ND provides a novel means to investigate biochemical aspects of CoQ10 uptake by cells and/or administer it to subjects deficient in this key lipid as a result of inborn errors of metabolism, statin therapy, or otherwise.