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Severely premature infants are often at increased risk of cerebral hemorrhage and/or ischemic injury caused by immature autoregulatory control of blood flow to the brain. If blood flow is too high, the infant is at risk of hemorrhage, whereas too little blood flow can result in ischemic injury. The development of a noninvasive, bedside means of measuring cerebral hemodynamics would greatly facilitate both diagnosis and monitoring of afflicted individuals. It is to this end that we have developed a near infrared spectroscopy (NIRS) system that allows for quantitative, bedside measurement of cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT). The technique requires an i.v. injection of the near infrared chromophore indocyanine green. Six newborn piglets, median age of 18 h (range 6 -54 h), median weight of 1.75 kg (range 1.5-2.1 kg), were studied. Measurements of CBF, CBV, and MTT were made at normocapnia, hypocapnia, and hypercapnia to test the technique over a range of hemodynamic conditions. The accuracy of our new approach has been determined by direct comparison with measurements made using a previously validated computed tomography technique. Paired t tests showed no significant difference between computed tomography and NIRS measurements of CBF, CBV, and MTT, and mean biases between the two methods were Ϫ2.05 mL·min Ϫ1 ·100 g Ϫ1 , Ϫ0.18 mL·100 g Ϫ1 , and 0.43 s, respectively. The precision of NIRS CBF, CBV, and MTT measurements, as determined by repeatedmeasures ANOVA, was 9.71%, 13.05%, and 7.57%, respectively. Since the first publication by Jobsis in 1977 (1), NIRS has been used in a variety of studies to investigate cerebral hemodynamics (2, 3). The underlying principles behind the use of NIRS to probe biologic media are relatively simple and have been described in detail elsewhere (1, 4 -8). There exist in biologic tissue four endogenous near infrared (NIR) light absorbers-oxy-Hb (HbO 2 ), deoxy-Hb (Hb), cytochrome oxidase (Cyt), and water. Because HbO 2 and Hb are generally present at relatively low concentrations in tissue, NIR light is able to penetrate tissue to a greater extent than other low-energy forms of light, in some cases up to distances of 8 -9 cm (6).As NIR light enters tissue, it is multiply scattered. The result of this scatter is that the total path length traveled by the NIR light is greater than the physical distance between the points of emission and detection. This extra distance can be accounted for using the differential path length factor (DPF), first described by Delpy et al. (9). With accurate knowledge of the DPF, a modified version of the Beer-Lambert law can be used to determine absolute changes in concentrations of NIR absorbers within tissue:where ⌬c is the change in concentration, ⌬A is the change in attenuation, ␣ is the extinction coefficient, L is the physical distance between emission and detection of NIR light, and B is the DPF. Measurement of concentration changes over time can

A multichannel spectrally resolved optical tomography system to image molecular targets in small animals from within a clinical MRI is described. Long source/detector fibers operate in contact mode and couple light from the tissue surface in the magnet bore to 16 spectrometers, each containing two optical gratings optimized for the near infrared wavelength range. High sensitivity, cooled charge coupled devices connected to each spectrograph provide detection of the spectrally resolved signal, with exposure times that are automated for acquisition at each fiber. The design allows spectral fitting of the remission light, thereby separating the fluorescence signal from the nonspecific background, which improves the accuracy and sensitivity when imaging low fluorophore concentrations. Images of fluorescence yield are recovered using a nonlinear reconstruction approach based on the diffusion approximation of photon propagation in tissue. The tissue morphology derived from the MR images serves as an imaging template to guide the optical reconstruction algorithm. Sensitivity studies show that recovered values of indocyanine green fluorescence yield are linear to concentrations of 1 nM in a 70 mm diameter homogeneous phantom, and detection is feasible to near 10 pM. Phantom data also demonstrate imaging capabilities of imperfect fluorophore uptake in tissue volumes of clinically relevant sizes. A unique rodent MR coil provides optical fiber access for simultaneous optical and MR data acquisition of small animals. A pilot murine study using an orthotopic glioma tumor model demonstrates optical-MRI imaging of an epidermal growth factor receptor targeted fluorescent probe in vivo.

The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, providing key steps in oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α--helices linked with a loop and short α--helix on the matrix side. The transporter cycles between a cytoplasmic and matrix conformation in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic conformation, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix conformation. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. The simultaneous 2 rotation of three domains around a central translocation pathway constitutes a unique mechanism among transport proteins.

We have investigated in whole cells whether, at low oxygen concentrations ([O 2]), endogenous nitric oxide (NO) modulates the redox state of the mitochondrial electron transport chain (ETC), and whether such an action has any signaling consequences. Using a polarographic-and-spectroscopic-coupled system, we monitored redox changes in the ETC cytochromes b H, cc1, and aa3 during cellular respiration. T he role of nitric oxide (NO) as a signaling molecule involved in the physiology of the cardiovascular, pulmonary, and nervous systems has been clearly demonstrated (1). It is widely accepted that many of the signaling consequences of NO are mediated through activation of the biochemical target soluble guanylyl cyclase (2). In the last decade, however, another potential target has emerged, namely cytochrome c oxidase (CcO, complex IV), the mitochondrial enzyme responsible for reduction of O 2 into water in the final stage of the electron transport chain (ETC). NO reversibly inhibits CcO by competing with O 2 for the binuclear binding site (3-5). Moreover, activation of endothelial NO synthase (NOS) by bradykinin in respiring endothelial cells results in a decrease in the rate of O 2 consumption (VO 2 ), an effect that can be reversed by an inhibitor of NOS (6). The high affinity of CcO for NO suggests that the nanomolar concentrations of NO generated in tissues under basal conditions may play a role in the regulation of VO 2 (7).We have recently developed a polarographic-and-spectroscopic-coupled system based on visible light spectroscopy (VLS) to monitor changes in the redox states of the mitochondrial ETC cytochromes during cellular respiration (8). Using this system, we have confirmed previous studies showing an early reduction of cytochrome c at low O 2 concentration [O 2 ], without a change in VO 2 (9). This phenomenon has been postulated to be part of a mechanism to maintain VO 2 at decreasing [O 2 ], although there is no consensus as to the factors that control this process (10, 11). Increased generation of mitochondrial reactive oxygen species (ROS) at low [O 2 ] has previously been linked to the activation of various adaptive signaling pathways (12). However, at present, the mechanism responsible for the observed increase in ROS at low [O 2 ] is not clear (see ref. 13 for review). One possibility is that NO, via its contribution to the early reduction of ETC cytochromes, favors the generation of ROS. We have therefore studied the effects of endogenous NO on the ETC redox state in endothelial and monocytic cells and the subsequent signaling consequences, specifically the release of ROS and the activation of NF-B (14). Materials and MethodsReagents. Hepes, N-dodecyl-␤-D-maltoside, cytochrome c (from bovine heart mitochondria), N,N,NЈ,NЈ tetramethyl-p-phenylenediamine, sodium ascorbate, and dihydroethidium (DHE) were purchased from Sigma;CcO Activity. Experiments with the purified enzyme (from bovine heart mitochondria) were carried out using the VLS system described previously (8). Briefly, the sample was p...

SUMMARYCytochrome oxidase is the terminal electron acceptor of the mitochondrial respiratory chain. It is responsible for the vast majority of oxygen consumption in the body and essential for the efficient generation of cellular ATP. The enzyme contains four redox active metal centres ; one of these, the binuclear Cu A centre, has a strong absorbance in the near-infrared that enables it to be detectable in i o by near-infrared spectroscopy. However, the fact that the concentration of this centre is less than 10 % of that of haemoglobin means that its detection is not a trivial matter.Unlike the case with deoxyhaemoglobin and oxyhaemoglobin, concentration changes of the total cytochrome oxidase protein occur very slowly (over days) and are therefore not easily detectable by nearinfrared spectroscopy. However, the copper centre rapidly accepts and donates an electron, and can thus change its redox state quickly ; this redox change is detectable by near-infrared spectroscopy. Many factors can affect the Cu A redox state in i o , but the most significant is likely to be the molecular oxygen concentration (at low oxygen tensions, electrons build up on Cu A as reduction of oxygen by the enzyme starts to limit the steady-state rate of electron transfer).The factors underlying haemoglobin oxygenation, deoxygenation and blood volume changes are, in general, well understood by the clinicians and physiologists who perform near-infrared spectroscopy measurements. In contrast, the factors that control the steady-state redox level of Cu A in cytochrome oxidase are still a matter of active debate, even amongst biochemists studying the isolated enzyme and mitochondria. Coupled with the difficulties of accurate in i o measurements it is perhaps not surprising that the field of cytochrome oxidase near-infrared spectroscopy has a somewhat chequered past. Too often papers have been written with insufficient information to enable the measurements to be repeated and few attempts have been made to test the algorithms in i o.In recent years a number of research groups and commercial spectrometer manufacturers have made a concerted attempt to not only say how they are attempting to measure cytochrome oxidase by nearinfrared spectroscopy but also to demonstrate that they are really doing so. We applaud these attempts, which in general fall into three areas : first, modelling of data can be performed to determine what problems are likely to derail cytochrome oxidase detection algorithms (Matcher et al. 1995) ; secondly haemoglobin concentration changes can be made by haemodilution (using saline or artificial blood substitutes) in animals (Tamura 1993) or patients (Skov & Greisen 1994) ; and thirdly, the cytochrome oxidase redox state can be fixed by the use of mitochondrial inhibitors and then attempts made to cause spurious cytochrome changes by dramatically varying haemoglobin oxygenation, haemoglobin concentration and light scattering (Cooper et al. 1997).We have previously written reviews covering the difficulties of measuring the cyt...

Mitochondrial ADP/ATP carriers catalyze the equimolar exchange of ADP and ATP across the mitochondrial inner membrane. Structurally, they consist of three homologous domains with a single substrate binding site. They alternate between a cytoplasmic and matrix state in which the binding site is accessible to these compartments for binding of ADP or ATP. It has been proposed that cycling between states occurs by disruption and formation of a matrix and cytoplasmic salt bridge network in an alternating way, but formation of the latter has not been shown experimentally. Here, we show that state-dependent formation of the cytoplasmic salt bridge network can be demonstrated by measuring the effect of mutations on the thermal stability of detergent-solubilized carriers locked in a specific state. For this purpose, mutations were made to increase or decrease the overall interaction energy of the cytoplasmic network. When locked in the cytoplasmic state by the inhibitor carboxyatractyloside, the thermostabilities of the mutant and wild-type carriers were similar, but when locked in the matrix state by the inhibitor bongkrekic acid, they correlated with the predicted interaction energy of the cytoplasmic network, demonstrating its formation. Changing the interaction energy of the cytoplasmic network also had a profound effect on the kinetics of transport, indicating that formation of the network is a key step in the transport cycle. These results are consistent with a unique alternating access mechanism that involves the simultaneous rotation of the three domains around a central translocation pathway.

The use of near-infrared spectroscopy to measure noninvasively changes in the redox state of cerebral cytochrome oxidase in vivo is controversial. We therefore tested these measurements using a multiwavelength detector in the neonatal pig brain. Exchange transfusion with perfluorocarbons revealed that the spectrum of cytochrome oxidase in the near-infrared was identical in the neonatal pig, the adult rat, and in the purified enzyme. Under normoxic conditions, the neonatal pig brain contained 15 micromol/L deoxyhemoglobin, 29 micromol/L oxyhemoglobin, and 1.2 micromol/L oxidized cytochrome oxidase. The mitochondrial inhibitor cyanide was used to determine whether redox changes in cytochrome oxidase could be detected in the presence of the larger cerebral hemoglobin concentration. Addition of cyanide induced full reduction of cytochrome oxidase in both blooded and bloodless animals. In the blooded animals, subsequent anoxia caused large changes in hemoglobin oxygenation and concentration but did not affect the cytochrome oxidase near-infrared signal. Simultaneous blood oxygenation level-dependent magnetic resonance imaging measurements showed a good correlation with near-infrared measurements of deoxyhemoglobin concentration. Possible interference in the near-infrared measurements from light scattering changes was discounted by simultaneous measurements of the optical pathlength using the cerebral water absorbance as a standard chromophore. We conclude that, under these conditions, near-infrared spectroscopy can accurately measure changes in the cerebral cytochrome oxidase redox state.