The approach to crystallize membrane proteins as complexes with specific antibody fragments appears to be of general importance. The structure of the yeast cytochrome bc(1) complex reveals in detail the binding sites of the natural substrate coenzyme Q6 and the inhibitor stigmatellin. Buried water molecules close to the binding sites suggest possible pathways for proton uptake and release. A comparison with other cytochrome bc(1) complexes shows features that are specific to yeast.

During mouse embryonic development, neural progenitors lengthen the G1 phase of the cell cycle and this has been suggested to be a cause, rather than a consequence, of neurogenesis. To investigate whether G1 lengthening alone may cause the switch of cortical progenitors from proliferation to neurogenesis, we manipulated the expression of cdk/cyclin complexes and found that cdk4/cyclinD1 overexpression prevents G1 lengthening without affecting cell growth, cleavage plane, or cell cycle synchrony with interkinetic nuclear migration. Specifically, overexpression of cdk4/cyclinD1 inhibited neurogenesis while increasing the generation and expansion of basal (intermediate) progenitors, resulting in a thicker subventricular zone and larger surface area of the postnatal cortex originating from cdk4/cyclinD1-transfected progenitors. Conversely, lengthening of G1 by cdk4/cyclinD1-RNAi displayed the opposite effects. Thus, G1 lengthening is necessary and sufficient to switch neural progenitors to neurogenesis, and overexpression of cdk4/cyclinD1 can be used to increase progenitor expansion and, perhaps, cortical surface area.

Small diffusible redox proteins facilitate electron transfer in respiration and photosynthesis by alternately binding to integral membrane proteins. Specific and transient complexes need to be formed between the redox partners to ensure fast turnover. In respiration, the mobile electron carrier cytochrome c shuttles electrons from the cytochrome bc 1 complex to cytochrome c oxidase. Despite extensive studies of this fundamental step of energy metabolism, the structures of the respective electron transfer complexes were not known. Here we present the crystal structure of the complex between cytochrome c and the cytochrome bc 1 complex from Saccharomyces cerevisiae. The complex was crystallized with the help of an antibody fragment, and its structure was determined at 2.97-Å resolution. Cytochrome c is bound to subunit cytochrome c 1 of the enzyme. The tight and specific interactions critical for electron transfer are mediated mainly by nonpolar forces. The close spatial arrangement of the c-type hemes unexpectedly suggests a direct and rapid heme-to-heme electron transfer at a calculated rate of up to 8.3 ؋ 10 6 s ؊1 . Remarkably, cytochrome c binds to only one recognition site of the homodimeric multisubunit complex. Interestingly, the occupancy of quinone in the Qi site is higher in the monomer with bound cytochrome c, suggesting a coordinated binding and reduction of both electron-accepting substrates. Obviously, cytochrome c reduction by the cytochrome bc 1 complex can be regulated in response to respiratory conditions.

Biochemical data have shown that speci®c, tightly bound phospholipids are essential for activity of the cytochrome bc 1 complex (QCR), an integral membrane protein of the respiratory chain. However, the structure and function of such phospholipids are not yet known. Here we describe ®ve phospholipid molecules and one detergent molecule in the X-ray structure of yeast QCR at 2.3 A Ê resolution. Their individual binding sites suggest speci®c roles in facilitating structural and functional integrity of the enzyme. Interestingly, a phosphatidylinositol molecule is bound in an unusual interhelical position near the¯exible linker region of the Rieske iron±sulfur protein. Two possible proton uptake pathways at the ubiquinone reduction site have been identi®ed: the E/R and the CL/K pathway. Remarkably, cardiolipin is positioned at the entrance to the latter. We propose that cardiolipin ensures structural integrity of the proton-conducting protein environment and takes part directly in proton uptake. Site-directed mutagenesis of ligating residues con®rmed the importance of the phosphatidylinositol-and cardiolipin-binding sites. Keywords: cardiolipin/cytochrome bc 1 complex/ oxidoreductase/phospholipid/proton transfer IntroductionBiological membranes are essential for life. They provide a permeability barrier for cells and cell organelles and they form the matrix for membrane-spanning proteins. A general property of the phospholipid bilayer is to hinder diffusion of ions, a prerequisite for the generation of electrochemical potentials that are utilized for synthesis of ATP or active transport. The proportion of putative membrane proteins predicted from sequenced genomes is between 20 and 35% (Stevens and Arkin, 2000). The large number of membrane-embedded proteins covers a broad range of functions in cellular metabolism. A tight interaction of these molecules with the phospholipid bilayer is required to maintain the diffusion barrier. During the past few years, a number of structures of integral membrane proteins at high atomic resolution have emerged. Some of them show protein-associated phospholipid molecules (Fyfe et al., 2001). Biochemical studies have shown that phospholipids are essential for the activity of several membrane proteins (Dowhan, 1997), but the role of structurally resolved phospholipids has not been clear up to now and has not yet been addressed by site-directed mutagenesis (Fyfe et al., 2001). Elucidating the function of speci®cally bound phospholipid molecules might be essential to understand fully the molecular mechanism of a membrane protein.The ubiquinol:cytochrome c oxidoreductase (QCR; cytochrome bc 1 complex, EC 1.10.2.2) is a multisubunit membrane protein complex, which is one of the fundamental components of the respiratory and photosynthetic electron transfer chains. The enzyme catalyzes electron transfer from ubiquinol to cytochrome c and couples this process to electrogenic translocation of protons across the membrane Berry et al., 2000). Each monomer of the homodimeric complex contains th...

At first sight, the nervous and vascular systems seem to share little in common. However, neural and vascular cells not only are anatomically closely tied to each other, but they also utilize and respond to similar classes of signals to establish correct connectivity and wiring of their networks. Recent studies further provide evidence that this neurovascular crosstalk is more important for understanding the molecular basis of neurological disease than originally anticipated. Moreover, neurovascular strategies offer novel therapeutic opportunities for neurodegenerative disorders.

Brain function critically relies on blood vessels to supply oxygen and nutrients, to establish a barrier for neurotoxic substances, and to clear waste products. The archetypal vascular endothelial growth factor, VEGF, arose in evolution as a signal affecting neural cells, but was later co-opted by blood vessels to regulate vascular function. Consequently, VEGF represents an attractive target to modulate brain function at the neurovascular interface. On the one hand, VEGF is neuroprotective, through direct effects on neural cells and their progenitors and indirect effects on brain perfusion. In accordance, preclinical studies show beneficial effects of VEGF administration in neurodegenerative diseases, peripheral neuropathies and epilepsy. On the other hand, pathologically elevated VEGF levels enhance vessel permeability and leakage, and disrupt blood-brain barrier integrity, as in demyelinating diseases, for which blockade of VEGF may be beneficial. Here, we summarize current knowledge on the role and therapeutic potential of VEGF in neurological diseases.

P ercutaneous revascularization is an established treatment for femoro-popliteal artery disease.1 Yet, restenosis, reocclusion, and ensuing symptom recurrence can occur in as many as 50% of patients undergoing percutaneous transluminal angioplasty (PTA), often requiring repeat percutaneous or surgical intervention.2,3 The superficial femoral artery represents a unique challenging vessel.4 Bare-metal stents reduce restenosis versus PTA and have gained widespread adoption. 5,6 Alternative therapies, such as drug-eluting balloon (DEB), may be valuable and worthwhile considering their promise and evidence to achieve patency outcomes at least similar to stents but with nothing left behind. 7Several studies in coronary artery disease indicate effective restenosis inhibition by DEB in the treatment of in-stent restenosis whereas drug-eluting stent are the preferred medicated devices in most coronary de novo lesions. Two published randomized trials have so far provided favorable data on the usage of DEB versus PTA for the treatment of femoropopliteal arterial disease. 8,9 More recently, the effect of a novel paclitaxel coating formulation with urea excipient, a naturally occurring highly biocompatible hydrophilic component (FreePac, Medtronic, Santa Rosa, CA) was investigated within a multicenter registry in patients affected by femoral-popliteal arterial disease. 10 To reach a more in-depth understanding onBackground-Peripheral percutaneous transluminal angioplasty is fraught with a substantial risk of restenosis and reintervention.A drug-eluting balloon (DEB) based on a novel coating was compared with uncoated balloons in patients undergoing femoropopliteal percutaneous transluminal angioplasty. Methods and Results-Patients with symptomatic femoro-popliteal atherosclerotic disease undergoing percutaneous transluminalangioplasty were randomized to paclitaxel-coated IN.PACT Pacific or uncoated Pacific balloons. The primary end point was late lumen loss at 6 months assessed by blinded angiographic corelab quantitative analyses. Secondary end points were binary restenosis and Rutherford class change at 6 months, and target lesion revascularization plus major adverse clinical events (major adverse events=death, target limb amputation, or target lesion revascularization) at 6 and 12 months. Eighty-five patients (91 cases=interventional procedures) were randomized in 3 hospitals (44 to DEB and 47 to uncoated balloons). Average lesion length was 7.0±5.3 and 6.6±5.5cm for DEB and control arm, respectively.

An improved method for the electrotransformation of wild-type Corynebacterium glutamicum (ATCC 13032) is described. The two crucial alterations to previously developed methods are: cultivation of cells used for electrotransformation at 18 degrees C instead of 30 degrees C, and application of a heat shock immediately following electrotransformation. Cells cultivated at sub optimal temperature have a 100-fold improved transformation efficiency (10(8) cfu micrograms-1) for syngeneic DNA (DNA isolated from the same species). A heat shock applied to these cells following electroporation improved the transformation efficiency for xenogeneic DNA (DNA isolated from a different species). In combination, low cultivation temperature and heat shock act synergistically and increased the transformation efficiency by four orders of magnitude to 2.5 x 10(6) cfu micrograms-1 xenogeneic DNA. The method was used to generate gene disruptions in C. glutamicum.