Cutaneous respirometry by dynamic measurement of mitochondrial oxygen tension for monitoring mitochondrial function in vivo

Progress in diagnosis and treatment of mitochondrial dysfunction in chronic and acute disease could greatly benefit from techniques for monitoring of mitochondrial function in vivo. In this study we demonstrate the feasibility of in vivo respirometry in skin. Mitochondrial oxygen measurements by means of oxygen-dependent delayed fluorescence of protoporphyrin IX are shown to provide a robust basis for measurement of local oxygen disappearance rate (ODR). The fundamental principles behind the technology are described, together with an analysis method for retrievel of respirometry data. The feasibility and reproducibility of this clinically useful approach are demonstrated in a series of rats.     

Cortical Morphology Differences in Subjects at Increased Vulnerability for Developing a Psychotic Disorder: A Comparison between Subjects with Ultra-High Risk and 22q11.2 Deletion Syndrome

IntroductionSubjects with 22q11.2 deletion syndrome (22q11DS) and subjects with ultra-high risk for psychosis (UHR) share a risk of approximately 30% to develop a psychotic disorder. Studying these groups helps identify biological markers of pathophysiological processes involved in the development of psychosis. Total cortical surface area (cSA), total cortical grey matter volume (cGMV), cortical thickness (CT), and local gyrification index (LGI) of the cortical structure have a distinct neurodevelopmental origin making them important target markers to study in relation to the development of psychosis.Results22q11DS subjects had lower total cSA and total cGMV compared to UHR and HC subjects. The 22q11DS subjects showed bilateral lower LGI in the i) prefrontal cortex, ii) precuneus, iii) precentral gyrus and iv) cuneus compared to UHR subjects. Additionally, lower LGI was found in the left i) fusiform gyrus and right i) pars opercularis, ii) superior, and iii) inferior temporal gyrus in 22q11DS subjects compared to HC. In comparison to 22q11DS subjects, the UHR subjects had lower CT of the insula. For both risk groups, positive symptom severity was negatively correlated to rostral middle frontal gyrus CT.ConclusionA shared negative correlation between positive symptom severity and rostral middle frontal gyrus CT in UHR and 22q11DS may be related to their increased vulnerability to develop a psychotic disorder. 22q11DS subjects were characterised by widespread lower degree of cortical gyrification linked to early and postnatal neurodevelopmental pathology. No implications for early neurodevelopmental pathology were found for the UHR subjects, although they did have distinctively lower insula CT which may have arisen from defective pruning processes during adolescence. Implications of these findings in relation to development of psychotic disorders are in need of further investigation in longitudinal studies.     

Unique residues involved in activation of the multitasking protease/chaperone HtrA from Chlamydia trachomatis

DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrates of Chlamydia trachomatis HtrA (DegP homolog). We have demonstrated that CtHtrA proteolysis could be activated by allosteric binding and oligomer formation. The PDZ1 activator cleft was required for the activation and oligomer formation. However, unique to CtHtrA was the critical role for residues at the PDZ1:protease interface in oligomer formation when the activator was an in vitro chaperone substrate. Furthermore, a potential in vivo chaperone substrate, the major outer membrane protein (MOMP) from Chlamydia, was able to activate CtHtrA and induce oligomer formation. Therefore, we have revealed novel residues involved in the activation of CtHtrA which are likely to have important in vivo implications for outer membrane protein assembly.     

Heterologous overexpression and purification of cytochrome c' from Rhodobacter capsulatus and a mutant (K42E) in the dimerization region. Mutation does not alter oligomerization but impacts the heme iron spin state and nitric oxide binding properties

Rhodobacter capsulatus cytochrome c' (RCCP) has been overexpressed in Escherichia coli, and its spectroscopic and ligand-binding properties have been investigated. It is concluded that the heterologously expressed protein is assembled correctly, as judged by UV-vis absorption, EPR, and resonance Raman (RR) spectroscopy of the unligated protein as well as forms in which the heme is ligated by CO or NO. To probe the oligomerization state of RCCP and its potential influence on heme reactivity, we have compared the properties of wild-type RCCP with a mutant (K42E) that lacks a salt bridge at the subunit interface. Analytical ultracentrifugation indicates that wild-type and K42E proteins are both monomeric in solution, contrary to the homodimeric structure of the crystalline state. Surprisingly, the K42E mutation produces a number of changes at the heme center (nearly 20 A distant), including perturbation of the ferric spin-state equilibrium and a change in the ferrous heme-nitrosyl complex from a six-coordinate/five-coordinate mixture to a predominantly five-coordinate heme-NO species. RR spectra indicate that ferrous K42E and wild-type RCCP both have relatively high Fe-His stretching frequencies, suggesting that the more favored five-coordinate heme-nitrosyl formation in K42E is not caused by a weaker Fe2+-His bond. Nevertheless, the altered reactivity of ferrous K42E with NO, together with its modified ferric spin state, shows that structural changes originating at the dimer interface can affect the properties of the heme center, raising the exciting possibility that intermolecular encounters at the protein surface might modulate the reactivity of cytochrome c' in vivo.     

the active site residue V266 of Chlamydial HtrA is critical for substrate binding during both in vitro and in vivo conditions

HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1-S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding.     

Arabidopsis XXT5 gene encodes a putative alpha-1,6-xylosyltransferase that is involved in xyloglucan biosynthesis

The function of a putative xyloglucan xylosyltransferase from Arabidopsis thaliana (At1g74380; XXT5) was studied. The XXT5 gene is expressed in all plant tissues, with higher levels of expression in roots, stems and cauline leaves. A T-DNA insertion in the XXT5 gene generates a readily visible root hair phenotype (root hairs are shorter and form bubble-like extrusions at the tip), and also causes the alteration of the main root cellular morphology. Biochemical characterization of cell wall polysaccharides isolated from xxt5 mutant seedlings demonstrated decreased xyloglucan quantity and reduced glucan backbone substitution with xylosyl residues. Immunohistochemical analyses of xxt5 plants revealed a selective decrease in some xyloglucan epitopes, whereas the distribution patterns of epitopes characteristic for other cell wall polysaccharides remained undisturbed. Transformation of xxt5 plants with a 35S::HA-XXT5 construct resulted in complementation of the morphological, biochemical and immunological phenotypes, restoring xyloglucan content and composition to wild-type levels. These data provide evidence that XXT5 is a xyloglucan alpha-1,6-xylosyltransferase, and functions in the biosynthesis of xyloglucan.     

Brca2 (XRCC11) Deficiency Results in Radioresistant DNA Synthesis and a Higher Frequency of Spontaneous Deletions

We show here that the radiosensitive Chinese hamster cell mutant (V-C8) of group XRCC11 is defective in the breast cancer susceptibility gene Brca2. The very complex phenotype of V-C8 cells is complemented by a single human chromosome 13 providing the BRCA2 gene, as well as by the murine Brca2 gene. The Brca2 deficiency in V-C8 cells causes hypersensitivity to various DNA-damaging agents with an extreme sensitivity toward interstrand DNA cross-linking agents. Furthermore, V-C8 cells show radioresistant DNA synthesis after ionizing radiation, suggesting that Brca2 deficiency affects cell cycle checkpoint regulation. In addition, V-C8 cells display tremendous chromosomal instability and a high frequency of abnormal centrosomes. The mutation spectrum at the hprt locus showed that the majority of spontaneous mutations in V-C8 cells are deletions, in contrast to wild-type V79 cells. A mechanistic explanation for the genome instability phenotype of Brca2-deficient cells is provided by the observation that the nuclear localization of the central DNA repair protein in homologous recombination, Rad51, is reduced in V-C8 cells.     

Effects of Furosemide on Cochlear Neural Activity,Central Hyperactivity and Behavioural Tinnitus after Cochlear Trauma in Guinea Pig

Cochlear trauma causes increased spontaneous activity (hyperactivity) to develop in central auditory structures, and this has been suggested as a neural substrate for tinnitus. Using a guinea pig model we have previously demonstrated that for some time after cochlear trauma, central hyperactivity is dependent on peripheral afferent drive and only later becomes generated intrinsically within central structures. Furosemide, a loop diuretic, reduces spontaneous firing of auditory afferents. We investigated in our guinea pig model the efficacy of furosemide in reducing 1) spontaneous firing of auditory afferents, using the spectrum of neural noise (SNN) from round window recording, 2) hyperactivity in inferior colliculus, using extracellular single neuron recordings and 3) tinnitus at early time-points after cochlear trauma. Tinnitus was assessed using gap prepulse inhibition of acoustic startle (GPIAS). Intraperitoneal furosemide, but not saline, caused a marked decrease in both SNN and central hyperactivity. Intracochlear perfusion with furosemide similarly reversed central hyperactivity. In animals in which GPIAS measurements suggested the presence of tinnitus (reduced GPIAS), this could be reversed with an intraperitoneal injection with furosemide but not saline. The results are consistent with furosemide reducing central hyperactivity and behavioural signs of tinnitus by acting peripherally to decrease spontaneous firing of auditory afferents. The data support the notion that hyperactivity may be involved in the generation of tinnitus and further suggest that there may be a therapeutic window after cochlear trauma using drug treatments that target peripheral spontaneous activity.