Effect of the point mutation H148G on GFPmut2 unfolding kinetics by fluorescence spectroscopy

We have used fluorescence spectroscopy techniques such as fluorescence correlation spectroscopy and fluorescence anisotropy decay on a wide time range, from nanoseconds to seconds, to investigate the unfolding kinetics induced by guanidinium chloride of GFPMut2 and its point mutation H148G, which has proved to be relevant for GFP photochemistry and photophysics. The mutation affects the unfolding kinetics of GFP leading to a much faster process at alkaline pH values, where protonation dynamics is negligible, that can be ascribed to a twofold role of His148, either as a proton shutter towards the chromophore and as a conformation stabiliser. For both mutants a soft region located near beta-strand 3 is found that starts to gain flexibility in the ns range at denaturant concentrations far lower than those required to turn off the chromophore fluorescence, as derived from the anisotropy decay of an extrinsic probe covalently bound to the proteins.     

Cytochrome P-450 3A13 and endothelin jointly mediate ductus arteriosus constriction to oxygen in mice

The fetal ductus arteriosus (DA) contracts to oxygen, and this feature, maturing through gestation, is considered important for its closure at birth. We have previously obtained evidence of the involvement of cytochrome P-450, possibly of the 3A subfamily (CYP3A), in oxygen sensing and have also identified endothelin (ET)-1 as the attendant effector for the contraction. Here, we examined comparatively wild-type (WT) and CYP3A-null (Cyp3a(-/-)) mice for direct validation of this concept. We found that the CYP3A subfamily is represented only by CYP3A13 in the WT DA. CYP3A13 was also detected in the DA by immunofluorescence microscopy, being primarily colocalized with the endoplasmic reticulum in both endothelial and muscle cells. However, a distinct signal was also evident in the plasma membrane. Isolated DAs from term WT animals developed a sustained contraction to oxygen with transient contractions superimposed. Conversely, no tonic response occurred in Cyp3a(-/-) DAs, whereas the phasic response persisted unabated. Oxygen did not contract the preterm WT DA but caused a full-fledged contraction after retinoic acid (RA) treatment. RA also promoted an oxygen contraction in the Cyp3a(-/-) DA. However, responses of RA-treated WT and Cyp3a(-/-) mice differed in that only the former abated with ET-1 suppression. This implies the existence of an alternative target for RA responsible for the oxygen-induced contraction in the absence of CYP3A13. In vivo, the DA was constricted in WT and Cyp3a(-/-) newborns, although with a tendency to be less narrowed in the mutant. We conclude that oxygen acts primarily through the complex CYP3A13 (sensor)/ET-1 (effector) and, in an accessory way, directly onto ET-1. However, even in the absence of CYP3A13, the DA may close postnatally thanks to the contribution of ET-1 and the likely involvement of compensating mechanism(s) identifiable with an alternative oxygen-sensing system and/or the withdrawal of relaxing influence(s) operating prenatally.     

Mitochondrial dysfunction and effect of antiglycolytic bromopyruvic acid in GL15 glioblastoma cells

Most cancer cells, including GL15 glioblastoma cells, rely on glycolysis for energy supply. The effect of antiglycolytic bromopyruvate on respiratory parameters and viability of GL15 cells was investigated. Bromopyruvate caused Δψ_m and MTT collapse, ATP decrease, and cell viability loss without involving apoptotic or necrotic pathways. The autophagy marker LC3-II was increased. Δψ_m decrease was accompanied by reactive oxygen species (ROS) increase and cytochrome c (cyt c) disappearance, suggesting a link between free radical generation and intramitochondrial cyt c degradation. Indeed, the free radical inducer menadione caused a decrease in cyt c that was reversed by N-acetylcysteine. Cyt c is tightly bound to the inner mitochondrial membrane in GL15 cells, which may confer protein peroxidase activity, resulting in auto-oxidation and protein targeting to degradation in the presence of ROS. This process is directed towards impairment of the apoptotic cyt c cascade, although cells are committed to die.     

Functional impairment of skeletal muscle oxidative metabolism during knee extension exercise after bed rest

A functional evaluation of skeletal muscle oxidative metabolism during dynamic knee extension (KE) incremental exercises was carried out following a 35-day bed rest (BR) (Valdoltra 2008 BR campaign). Nine young male volunteers (age: 23.5 ± 2.2 yr; mean ± SD) were evaluated. Pulmonary gas exchange, heart rate and cardiac output (by impedance cardiography), skeletal muscle (vastus lateralis) fractional O(2) extraction, and brain (frontal cortex) oxygenation (by near-infrared spectroscopy) were determined during incremental KE. Values at exhaustion were considered "peak". Peak heart rate (147 ± 18 beats/min before vs. 146 ± 17 beats/min after BR) and peak cardiac output (17.8 ± 3.3 l/min before vs. 16.1 ± 1.8 l/min after BR) were unaffected by BR. As expected, brain oxygenation did not decrease during KE. Peak O(2) uptake was lower after vs. before BR, both when expressed as liters per minute (0.99 ± 0.17 vs. 1.26 ± 0.27) and when normalized per unit of quadriceps muscle mass (46.5 ± 6.4 vs. 56.9 ± 11.0 ml·min(-1)·100 g(-1)). Skeletal muscle peak fractional O(2) extraction, expressed as a percentage of the maximal values obtained during a transient limb ischemia, was lower after (46.3 ± 12.1%) vs. before BR (66.5 ± 11.2%). After elimination, by the adopted exercise protocol, of constraints related to cardiovascular O(2) delivery, a decrease in peak O(2) uptake and muscle peak capacity of fractional O(2) extraction was found after 35 days of BR. These findings suggest a substantial impairment of oxidative function at the muscle level, "downstream" with respect to bulk blood flow to the exercising muscles, that is possibly at the level of blood flow distribution/O(2) utilization inside the muscle, peripheral O(2) diffusion, and intracellular oxidative metabolism.     

Role of bifidobacteria in the activation of the lignan secoisolariciresinol diglucoside

Lignans are ubiquitous plant polyphenols, which have relevant health properties being the major phytoestrogens occurring in Western diets. Secoisolariciresinol (SECO) is the major dietary lignan mostly found in plants as secoisolariciresinol diglucoside (SDG). To exert biological activity, SDG requires being deglycosylated to SECO and transformed to enterodiol (ED) and enterolactone (EL) by the intestinal microbes. The involvement of bifidobacteria in the transformation of lignans glucosides has been investigated for the first time in this study. Twenty-eight strains were assayed for SDG and SECO activation. They all failed to transform SECO into reduced metabolites, excluding any role in ED and EL production. Ten Bifidobacterium cultures partially hydrolyzed SDG, giving both SECO and the monoglucoside with yields < 25%. When the cell-free extracts were assayed in SDG transformation, seven additional strains were active in the hydrolysis. Cellobiose induced β-glucosidase activity and caused the enhancement of both the rate of SDG hydrolysis and the final yield of SECO only in the strains capable of SDG bioconversion. The highest SDG conversion to SECO was achieved by Bifidobacterium pseudocatenulatum WC 401, which exhibited 75% yield in cellobiose-based medium after 48 h. These results indicate that SDG hydrolysis is not a common feature in Bifidobacterium genus, but selected probiotic strains can be combined to β-glucoside-based prebiotics to enhance the release of SECO, thus improving its bioavailability for absorption by colonic mucosa and/or the biotransformation to ED and EL by other intestinal microorganisms.     

Many facets of chromosome 3p cytogenetic findings in clear cell renal carcinoma: the need for agreement in assessment FISH analysis to avoid diagnostic errors

Abnormalities of the locus chromosome 3p and the entire chromosome 3 are involved in the cancerogenesis of clear cell renal carcinoma and may be detected by interphase fluorescence in situ hybridization (interphase FISH). We observed a variable detection rate of chromosome 3p/3 abnormalities in different series of clear cell renal carcinoma. Therefore, we focused on problematic issues when performing analysis on routinely available formalin-fixed and paraffin embedded tissue. A group of studies encountered a single approach to chromosome 3p detection, by using probe/s to map different codes of the short arm 3p without a control of the entire chromosome 3. Deletion of chromosome 3p and monosomy of chromosome 3 ranged from 38% to 100% in clear cell renal carcinoma. Cut-off values for the threshold were chosen randomly or obtained by calculation of the mean value plus 1 or 2 or 3 standard deviations. Loss of chromosome 3p was assessed either as the percentage of single signals on the total number of nuclei, or applying a double approach with corrections of control chromosome 3. Moreover, cut off values were sometimes arbitrarily corrected with the findings from normal adjacent renal parenchyma. A consensus of experts in the field is needed in order to define the best methodological approach and the appropriate threshold in assessment 3p deletion when interphase FISH is performed in clear cell renal carcinoma. This harbours relevant diagnostic and therapeutic implications, at light also of targeted therapies recently available to clear cell renal carcinoma.     

The conserved factor DE-ETIOLATED 1 cooperates with CUL4-DDB1DDB2 to maintain genome integrity upon UV stress

Plants and many other eukaryotes can make use of two major pathways to cope with mutagenic effects of light, photoreactivation and nucleotide excision repair (NER). While photoreactivation allows direct repair by photolyase enzymes using light energy, NER requires a stepwise mechanism with several protein complexes acting at the levels of lesion detection, DNA incision and resynthesis. Here we investigated the involvement in NER of DE-ETIOLATED 1 (DET1), an evolutionarily conserved factor that associates with components of the ubiquitylation machinery in plants and mammals and acts as a negative repressor of light-driven photomorphogenic development in Arabidopsis. Evidence is provided that plant DET1 acts with CULLIN4-based ubiquitin E3 ligase, and that appropriate dosage of DET1 protein is necessary for efficient removal of UV photoproducts through the NER pathway. Moreover, DET1 is required for CULLIN4-dependent targeted degradation of the UV-lesion recognition factor DDB2. Finally, DET1 protein is degraded concomitantly with DDB2 upon UV irradiation in a CUL4-dependent mechanism. Altogether, these data suggest that DET1 and DDB2 cooperate during the excision repair process.     

Differential regulation of the postsynaptic clustering of γ-aminobutyric acid type A (GABAA) receptors by collybistin isoforms

Collybistin promotes submembrane clustering of gephyrin and is essential for the postsynaptic localization of gephyrin and γ-aminobutyric acid type A (GABA(A)) receptors at GABAergic synapses in hippocampus and amygdala. Four collybistin isoforms are expressed in brain neurons; CB2 and CB3 differ in the C terminus and occur with and without the Src homology 3 (SH3) domain. We have found that in transfected hippocampal neurons, all collybistin isoforms (CB2(SH3+), CB2(SH3-), CB3(SH3+), and CB3(SH3-)) target to and concentrate at GABAergic postsynapses. Moreover, in non-transfected neurons, collybistin concentrates at GABAergic synapses. Hippocampal neurons co-transfected with CB2(SH3-) and gephyrin developed very large postsynaptic gephyrin and GABA(A) receptor clusters (superclusters). This effect was accompanied by a significant increase in the amplitude of miniature inhibitory postsynaptic currents. Co-transfection with CB2(SH3+) and gephyrin induced the formation of many (supernumerary) non-synaptic clusters. Transfection with gephyrin alone did not affect cluster number or size, but gephyrin potentiated the clustering effect of CB2(SH3-) or CB2(SH3+). Co-transfection with CB2(SH3-) or CB2(SH3+) and gephyrin did not affect the density of presynaptic GABAergic terminals contacting the transfected cells, indicating that collybistin is not synaptogenic. Nevertheless, the synaptic superclusters induced by CB2(SH3-) and gephyrin were accompanied by enlarged presynaptic GABAergic terminals. The enhanced clustering of gephyrin and GABA(A) receptors induced by collybistin isoforms was not accompanied by enhanced clustering of neuroligin 2. Moreover, during the development of GABAergic synapses, the clustering of gephyrin and GABA(A) receptors preceded the clustering of neuroligin 2. We propose a model in which the SH3- isoforms play a major role in the postsynaptic accumulation of GABA(A) receptors and in GABAergic synaptic strength.