Richard J. Shannon

Bovine Complex I Is a Complex of 45 Different Subunits

Mammalian mitochondrial complex I is a multisubunit membrane-bound assembly with a molecular mass approaching 1 MDa. By comprehensive analyses of the bovine complex and its constituent subcomplexes, 45 different subunits have been characterized previously. The presence of a 46th subunit was suspected from the consistent detection of a molecular mass of 10,566 by electrospray ionization mass spectrometry of subunits fractionated by reverse-phase high pressure liquid chromatography. The component was found associated with both the intact complex and subcomplex Iβ, which represents most of the membrane arm of the complex, and it could not be resolved chromatographically from subunit SGDH (the subunit of bovine complex I with the N-terminal sequence Ser-Gly-Asp-His). It has now been characterized by tandem mass spectrometry of intact protein ions and shown to be a C-terminal fragment of subunit SGDH arising from a specific peptide bond cleavage between Ile-55 and Pro-56 during the electrospray ionization process. Thus, the subunit composition of bovine complex I has been established. It is a complex of 45 different proteins plus non-covalently bound FMN and eight iron-sulfur clusters.

Analysis of the Subunit Composition of Complex I from Bovine Heart Mitochondria

Complex I purified from bovine heart mitochondria is a multisubunit membrane-bound assembly. In the past, seven of its subunits were shown to be products of the mitochondrial genome, and 35 nuclear encoded subunits were identified. The complex is L-shaped with one arm in the plane of the membrane and the other lying orthogonal to it in the mitochondrial matrix. With mildly chaotropic detergents, the intact complex has been resolved into various subcomplexes. Subcomplex Iλ represents the extrinsic arm, subcomplex Iα consists of subcomplex Iλ plus part of the membrane arm, and subcomplex Iβ is another substantial part of the membrane arm. The intact complex and these three subcomplexes have been subjected to extensive reanalysis. Their subunits have been separated by three independent methods (one-dimensional SDS-PAGE, two-dimensional isoelectric focusing/SDS-PAGE, and reverse phase high pressure liquid chromatography (HPLC)) and analyzed by tryptic peptide mass fingerprinting and tandem mass spectrometry. The masses of many of the intact subunits have also been measured by electrospray ionization mass spectrometry and have provided valuable information about post-translational modifications. The presence of the known 35 nuclear encoded subunits in complex I has been confirmed, and four additional nuclear encoded subunits have been detected. Subunits B16.6, B14.7, and ESSS were discovered in the SDS-PAGE analysis of subcomplex Iλ, in the two-dimensional gel analysis of the intact complex, and in the HPLC analysis of subcomplex Iβ, respectively. Despite many attempts, no sequence information has been obtained yet on a fourth new subunit (mass 10,566 ± 2 Da) also detected in the HPLC analysis of subcomplex Iβ. It is unlikely that any more subunits of the bovine complex remain undiscovered. Therefore, the intact enzyme is a complex of 46 subunits, and, assuming there is one copy of each subunit in the complex, its mass is 980 kDa.

The nuclear encoded subunits of complex I from bovine heart mitochondria.

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a complicated, multi-subunit, membrane-bound assembly. Recently, the subunit compositions of complex I and three of its subcomplexes have been reevaluated comprehensively. The subunits were fractionated by three independent methods, each based on a different property of the subunits. Forty-six different subunits, with a combined molecular mass of 980 kDa, were identified. The three subcomplexes, I alpha, I beta and I lambda, correlate with parts of the membrane extrinsic and membrane-bound domains of the complex. Therefore, the partitioning of subunits amongst these subcomplexes has provided information about their arrangement within the L-shaped structure. The sequences of 45 subunits of complex I have been determined. Seven of them are encoded by mitochondrial DNA, and 38 are products of the nuclear genome, imported into the mitochondrion from the cytoplasm. Post-translational modifications of many of the nuclear encoded subunits of complex I have been identified. The seven mitochondrially encoded subunits, and seven of the nuclear encoded subunits, are homologues of the 14 subunits found in prokaryotic complexes I. They are considered to be sufficient for energy transduction by complex I, and they are known as the core subunits. The core subunits bind a flavin mononucleotide (FMN) at the active site for NADH oxidation, up to eight iron-sulfur clusters, and one or more ubiquinone molecules. The locations of some of the cofactors can be inferred from the sequences of the core subunits. The remaining 31 subunits of bovine complex I are the supernumerary subunits, which may be important either for the stability of the complex, or for its assembly. Sequence relationships suggest that some of them carry out reactions unrelated to the NADH:ubiquinone oxidoreductase activity of the complex.

GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH: Ubiquinone oxidoreductase (complex I)

The sequences of 42 subunits of NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria have been described previously. Seven are encoded by mitochondrial DNA, whereas the remaining 35 are nuclear gene products imported into the organelle from the cytoplasm. An additional protein, which does not correspond to any previously known subunit of the complex I assembly, has now been detected. Denaturing gels of subcomplex Iλ, the hydrophilic arm of complex I, clearly show a hitherto unidentified band, which was digested with trypsin and subjected to mass-spectrometric analysis to provide several peptide sequences, used in cDNA cloning and sequencing. Measurement of the intact protein mass indicated that the N terminus is acetylated. The new complex I subunit (B16.6) is the bovine homolog of GRIM-19, the product of a cell death regulatory gene induced by interferon-β and retinoic acid, thus providing a new link between the mitochondrion and its electron-transport chain and apoptotic cell death.

Consensus statement from the 2014 International Microdialysis Forum

Microdialysis enables the chemistry of the extracellular interstitial space to be monitored. Use of this technique in patients with acute brain injury has increased our understanding of the pathophysiology of several acute neurological disorders. In 2004, a consensus document on the clinical application of cerebral microdialysis was published. Since then, there have been significant advances in the clinical use of microdialysis in neurocritical care. The objective of this review is to report on the International Microdialysis Forum held in Cambridge, UK, in April 2014 and to produce a revised and updated consensus statement about its clinical use including technique, data interpretation, relationship with outcome, role in guiding therapy in neurocritical care and research applications.

The Post-translational Modifications of the Nuclear Encoded Subunits of Complex I from Bovine Heart Mitochondria

Bovine complex I is an assembly of 46 different proteins. Seven of them are encoded in mitochondrial DNA, and the rest are nuclear gene products that are imported into the organelle. Fourteen of the nuclear encoded subunits have modified N termini. Many of these post-translational modifications have been deduced previously from intact protein masses. These assignments have been verified by mass spectrometric analysis of peptides. Thirteen of them are N-α-acetylated, and a 14th, subunit B18, is N-α-myristoylated. Subunit B18 forms part of the membrane arm of the complex, and the myristoyl group may attach subunit B18 to the membrane. One subunit, B12, has a particularly complex pattern of post-translational modification that has not been analyzed before. It is a mixture of the N-α-acetylated form and the form with a free N terminus. In addition, it has one, two, or three methyl groups attached to histidine residues at positions 4, 6, and 8 in various combinations. The predominant form is methylated on residues 4 and 6. There is no evidence for the methylation of histidine 2. Subunit B12 is also part of the membrane arm of complex I, and it probably spans the membrane once, but as its orientation is not known, the methylation sites could be in either the matrix or the intermembrane space. These experiments represent another significant step toward establishing the precise chemical composition of mammalian complex I.

Cerebral microdialysis in clinical studies of drugs: pharmacokinetic applications

The ability to deliver drug molecules effectively across the blood–brain barrier into the brain is important in the development of central nervous system (CNS) therapies. Cerebral microdialysis is the only existing technique for sampling molecules from the brain extracellular fluid (ECF; also termed interstitial fluid), the compartment to which the astrocytes and neurones are directly exposed. Plasma levels of drugs are often poor predictors of CNS activity. While cerebrospinal fluid (CSF) levels of drugs are often used as evidence of delivery of drug to brain, the CSF is a different compartment to the ECF. The continuous nature of microdialysis sampling of the ECF is ideal for pharmacokinetic (PK) studies, and can give valuable PK information of variations with time in drug concentrations of brain ECF versus plasma. The microdialysis technique needs careful calibration for relative recovery (extraction efficiency) of the drug if absolute quantification is required. Besides the drug, other molecules can be analysed in the microdialysates for information on downstream targets and/or energy metabolism in the brain. Cerebral microdialysis is an invasive technique, so is only useable in patients requiring neurocritical care, neurosurgery or brain biopsy. Application of results to wider patient populations, and to those with different pathologies or degrees of pathology, obviously demands caution. Nevertheless, microdialysis data can provide valuable guidelines for designing CNS therapies, and play an important role in small phase II clinical trials. In this review, we focus on the role of cerebral microdialysis in recent clinical studies of antimicrobial agents, drugs for tumour therapy, neuroprotective agents and anticonvulsants.

Directed macrocyclisation reactions

Macrocyclic hosts are prepared in 80–90% yields by using intramolecular hydrogen-bonding interactions to direct the cyclisation; in the absence of such effects, intermolecular hydrogen-bonding interactions template the formation of catenanes.

Monitoring vigabatrin in head injury patients by cerebral microdialysis: obtaining pharmacokinetic measurements in a neurocritical care setting

Aims The aims were to determine blood–brain barrier penetration and brain extracellular pharmacokinetics for the anticonvulsant vigabatrin (VGB; γ-vinyl-γ-aminobutyric acid) in brain extracellular fluid and plasma from severe traumatic brain injury (TBI) patients, and to measure the response of γ-aminobutyric acid (GABA) concentration in brain extracellular fluid. Methods Severe TBI patients (n = 10) received VGB (0.5 g enterally, every 12 h). Each patient had a cerebral microdialysis catheter; two patients had a second catheter in a different region of the brain. Plasma samples were collected 0.5 h before and 2, 4 and 11.5 h after the first VGB dose. Cerebral microdialysis commenced before the first VGB dose and continued through at least three doses of VGB. Controls were seven severe TBI patients with microdialysis, without VGB. Results After the first VGB dose, the maximum concentration of VGB (Cmax) was 31.7 (26.9–42.6) μmol l−1 (median and interquartile range for eight patients) in plasma and 2.41 (2.03–5.94) μmol l−1 in brain microdialysates (nine patients, 11 catheters), without significant plasma–brain correlation. After three doses, median Cmax in microdialysates increased to 5.22 (4.24–7.14) μmol l−1 (eight patients, 10 catheters). Microdialysate VGB concentrations were higher close to focal lesions than in distant sites. Microdialysate GABA concentrations increased modestly in some of the patients after VGB administration. Conclusions Vigabatrin, given enterally to severe TBI patients, crosses the blood–brain barrier into the brain extracellular fluid, where it accumulates with multiple dosing. Pharmacokinetics suggest delayed uptake from the blood.

Extracellular N-Acetylaspartate in Human Traumatic Brain Injury.

Abstract N-acetylaspartate (NAA) is an amino acid derivative primarily located in the neurons of the adult brain. The function of NAA is incompletely understood. Decrease in brain tissue NAA is presently considered symptomatic and a potential biomarker of acute and chronic neuropathological conditions. The aim of this study was to use microdialysis to investigate the behavior of extracellular NAA (eNAA) levels after traumatic brain injury (TBI). Sampling for this study was performed using cerebral microdialysis catheters (M Dialysis 71) perfused at 0.3 μL/min. Extracellular NAA was measured in microdialysates by high-performance liquid chromatography in 30 patients with severe TBI and for comparison, in radiographically “normal” areas of brain in six non-TBI neurosurgical patients. We established a detailed temporal eNAA profile in eight of the severe TBI patients. Microdialysate concentrations of glucose, lactate, pyruvate, glutamate, and glycerol were measured on an ISCUS clinical microdialysis analyzer. Here, we show that the temporal profile of microdialysate eNAA was characterized by highest levels in the earliest time-points post-injury, followed by a steady decline; beyond 70 h post-injury, average levels were 40% lower than those measured in non-TBI patients. There was a significant inverse correlation between concentrations of eNAA and pyruvate; eNAA showed significant positive correlations with glycerol and the lactate/pyruvate (L/P) ratio measured in microdialysates. The results of this on-going study suggest that changes in eNAA after TBI relate to the release of intracellular components, possibly due to neuronal death or injury, as well as to adverse brain energy metabolism.