Individual heme a and heme a3 contributions to the Soret absorption spectrum of the reduced bovine cytochrome c oxidase.

Bovine cytochrome c oxidase (CcO) contains two hemes, a and a3, chemically identical but differing in coordination and spin state. The Soret absorption band of reduced aa3-type cytochrome c oxidase consists of overlapping bands of the hemes a2+ and a32+. It shows a peak at ∼444 nm and a distinct shoulder at ∼425 nm. However, attribution of individual spectral lineshapes to hemes a2+ and a32+ in the Soret is controversial. In the present work, we characterized spectral contributions of hemes a2+ and a32+ using two approaches. First, we reconstructed bovine CcO heme a2+ spectrum using a selective Ca2+-induced spectral shift of the heme a2+. Second, we investigated photobleaching of the reduced Thermus thermophilus ba3- and bovine aa3-oxidases in the Soret induced by femtosecond laser pulses in the Q-band. The resolved spectra show splitting of the electronic B0x-, B0y-transitions of both reduced hemes. The heme a2+ spectrum is shifted to the red relative to heme a32+ spectrum. The ∼425 nm shoulder is mostly attributed to heme a32+.

CLARITY analysis of the Cl/pH sensor expression in the brain of transgenic mice.

Genetically encoded biosensors are widely used in cell biology for the non-invasive imaging of concentrations of ions or the activity of enzymes, to evaluate the distribution of small molecules, proteins and organelles, and to image protein interactions in living cells. These fluorescent molecules can be used either by transient expression in cultured cells or in entire organisms or through stable expression by producing transgenic animals characterized by genetically encoded and heritable biosensors. Using the mouse Thy1 mini-promoter, we generated a line of transgenic mice expressing a genetically encoded sensor for the simultaneous measurements of intracellular Cl- and pH. This construct, called ClopHensor, consists of a H+- and Cl--sensitive variant of the enhanced green fluorescent protein (E2GFP) fused with a red fluorescent protein (DsRedm). Stimulation of hippocampal Schaffer collaterals proved that the sensor is functionally active. To reveal the expression pattern of ClopHensor across the brain of Thy1::ClopHensor mice, we obtained transparent brain samples using the CLARITY method and imaged them with confocal and light-sheet microscopy. We then developed a semi-quantitative approach to identify brain structures with high intrinsic sensor fluorescence. This approach allowed us to assess cell morphology and track axonal projection, as well as to confirm E2GFP and DsRedm fluorescence colocalization. This analysis also provides a map of the brain areas suitable for non-invasive monitoring of intracellular Cl-/pH in normal and pathological conditions. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

The energetic brain - A review from students to students.

The past 20 years have resulted in unprecedented progress in understanding brain energy metabolism and its role in health and disease. In this review, which was initiated at the 14th International Society for Neurochemistry Advanced School, we address the basic concepts of brain energy metabolism and approach the question of why the brain has high energy expenditure. Our review illustrates that the vertebrate brain has a high need for energy because of the high number of neurons and the need to maintain a delicate interplay between energy metabolism, neurotransmission, and plasticity. Disturbances to the energetic balance, to mitochondria quality control or to glia-neuron metabolic interaction may lead to brain circuit malfunction or even severe disorders of the CNS. We cover neuronal energy consumption in neural transmission and basic ('housekeeping') cellular processes. Additionally, we describe the most common (glucose) and alternative sources of energy namely glutamate, lactate, ketone bodies, and medium chain fatty acids. We discuss the multifaceted role of non-neuronal cells in the transport of energy substrates from circulation (pericytes and astrocytes) and in the supply (astrocytes and microglia) and usage of different energy fuels. Finally, we address pathological consequences of disrupted energy homeostasis in the CNS.