Reactive oxygen species and permeability transition pore in rat liver and kidney mitoplasts

Mitochondrial permeability transition is typically characterized by Ca²⁺ and oxidative stress-induced opening of a nonselective proteinaceous membrane pore sensitive to cyclosporin A, known as the permeability transition pore (PTP). Data from our laboratory provide evidence that the PTP is formed when inner membrane proteins aggregate as a result of disulfide cross-linking caused by thiol oxidation. Here we compared the redox properties between PTP in intact mitochondria and mitoplasts. The rat liver mitoplasts retained less than 5% and 10% of the original outer membrane markers monoamine oxidase and VDAC, respectively. Kidney mitoplasts also showed a partial depletion of hexokinase. In line with the redox nature of the PTP, mitoplasts that were more susceptible to PTP opening than intact mitochondria showed higher rates of H₂O₂ generation and decreased matrix NADPH-dependent antioxidant activity. Mitoplast PTP was also sensitive to the permeability transition inducer tert-butyl hydroperoxide and to the inhibitors cyclosporin A, EGTA, ADP, dithiothreitol and catalase. Taken together, these data indicate that, in mitoplasts, PTP exhibits redox regulatory characteristics similar to those described for intact mitochondria.     

Epineurial Window Is More Efficient in Attracting Axons than Simple Coaptation in a Sutureless (Cyanoacrylate-Bound) Model of End-to-Side Nerve Repair in the Rat Upper Limb: Functional and Morphometric Evidences and Review of the Literature

End-to-side nerve coaptation brings regenerating axons from the donor to the recipient nerve. Several techniques have been used to perform coaptation: microsurgical sutures with and without opening a window into the epi(peri)neurial connective tissue; among these, window techniques have been proven more effective in inducing axonal regeneration. The authors developed a sutureless model of end-to-side coaptation in the rat upper limb. In 19 adult Wistar rats, the median and the ulnar nerves of the left arm were approached from the axillary region, the median nerve transected and the proximal stump sutured to the pectoral muscle to prevent regeneration. Animals were then randomly divided in two experimental groups (7 animals each, 5 animals acting as control): Group 1: the distal stump of the transected median nerve was fixed to the ulnar nerve by applying cyanoacrylate solution; Group 2: a small epineurial window was opened into the epineurium of the ulnar nerve, caring to avoid damage to the nerve fibres; the distal stump of the transected median nerve was then fixed to the ulnar nerve by applying cyanoacrylate solution. The grasping test for functional evaluation was repeated every 10–11 weeks starting from week-15, up to the sacrifice (week 36). At week 36, the animals were sacrificed and the regenerated nerves harvested and processed for morphological investigations (high-resolution light microscopy as well as stereological and morphometrical analysis). This study shows that a) cyanoacrylate in end-to-side coaptation produces scarless axon regeneration without toxic effects; b) axonal regeneration and myelination occur even without opening an epineurial window, but c) the window is related to a larger number of regenerating fibres, especially myelinated and mature, and better functional outcomes.     

Role of the duplicated CCAAT box region in gamma-globin gene regulation and hereditary persistence of fetal haemoglobin.

Hereditary persistence of fetal haemoglobin (HPFH) is a clinically important condition in which a change in the developmental specificity of the gamma-globin genes results in varying levels of expression of fetal haemoglobin in the adult. The condition is benign and can significantly alleviate the symptoms of thalassaemia or sickle cell anaemia when co-inherited with these disorders. We have examined structure-function relationships in the -117 HPFH gamma promoter by analysing the effect of mutating specific promoter elements on the functioning of the wild-type and HPFH promoters. We find that CCAAT box mutants dramatically affect expression from the HPFH promoter in adult blood but have little effect on embryonic/fetal expression from the wild-type promoter. Our results suggest that there are substantial differences in the structure of the wild-type gamma promoter expressed early in development and the adult HPFH promoter. Together with previous results, this suggests that gamma silencing is a complex multifactorial phenomenon rather than being the result of a simple repressor binding to the promoter. We present a model for gamma-globin gene silencing that has significant implications for attempts to reactivate the gamma promoters in human adults by pharmacological means.     

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to “ribosomal stress” with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis.     

Incomplete Coverage of Candidate Genes: A Poorly Considered Bias

Current genetic investigations are performed both on the basis of a rational and biologically based choice of candidate genes and through genome wide scans. Nonetheless, lack of replication is a common problem in psychiatric genetics as well as in other genetic fields. There are a number of reasons for this inconsistency, among them a well known but poorly considered issue is gene coverage. The aim of the present paper is to focus on this well known and defectively deemed bias, especially when a candidate gene approach is chosen. The rational and the technical feasibility of this proposal are discussed as well as a survey of current investigations. The known consistent methodology to fix this bias is also discussed.Key Words: Candidate genes, association studies, psychiatry genetics, methodology.     

Source strength increases with the increasing precociousness of fruit maturation in field-grown clones of conilon coffee (Coffea canephora) trees

In earlier-maturing coffee clones, owing to the shorter time required for fruit filling and ripening, photo-assimilates should be transported to fruits in a period shorter than that found in intermediate- or late-maturing clones. We hypothesised that at a given source-to-sink ratio, a presumably greater sink strength in early-maturing clones relative to intermediate- and late-maturing individuals should be correlated to increased rate of net carbon assimilation (A) and greater photo-assimilate transport to the fruits. Overall, earlier-maturing clones displayed greater A rates than the intermediate-maturing clones, which, in turn, had higher A than their late counterparts. Changes in A were largely associated with changes in stomatal conductance. Only marginal alterations occurred in the internal-to-ambient CO₂ concentration ratio, the carbon isotope composition ratio, soluble sugars and chlorophyll a fluorescence parameters. Some changes in starch pools were detected among treatments. To the best of our knowledge, this is the first report showing evidence that increased precociousness of fruit growth and maturation results in higher A and thus increased source strength, a fact associated to a large degree with higher stomatal aperture.     

Two dimensional non-equilibrium pH gel electrophoresis mapping of cytosolic protein changes caused by dietary protein depletion in mouse liver

Two-dimensional non-equilibrium pH gel electrophoresis (2D-NEPHGE) analysis was used to evaluate the effects of dietary protein depletion on the protein composition of mouse liver cytosol. Analysing the cytosol from both normal and protein depleted liver, the position in gels of more than three hundred protein spots was determined. After 5 days of protein depletion, about 20% of the spots either increased or decreased more than 2 fold. Five spots of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recognised by specific antibodies. The glutathione S-transferase (GSTs) subunits Yb1, Yc and Yf were identified by the simultaneous analysis of both glutathione-binding cytosolic proteins and the corresponding standards. As estimated by internal optical density (IOD) of spots, the changes caused by protein depletion in GAPDH and GST subunit contents were similar to those obtained by other methods. By means of mass spectrometric analysis of tryptic peptides generated from spots and/or comparison of two-dimensional gel electrophoretic patterns, carbonic anhydrase III (CAIII), Cu, Zn superoxide dismutase (CuZnSOD) and a cytochrome P450 cytosolic protein (cyt P450) were identified. These three proteins, as well as GSTs, are related with intracellular detoxification and free radical scavenging systems. Their contents were regulated by dietary protein restriction in a manner indicative of diminished liver defence against oxidising agents.     

Purification of beef kidney D-aspartate oxidase overexpressed in Escherichia coli and characterization of its redox potentials and oxidative activity towards agonists and antagonists of excitatory amino acid receptors

The flavoenzyme d-aspartate oxidase from beef kidney (DASPO, EC 1.4. 3.1) has been overexpressed in Escherichia coli. A purification procedure, faster than the one used for the enzyme from the natural source (bDASPO), has been set up yielding about 2 mg of pure recombinant protein (rDASPO) per each gram of wet E. coli paste. rDASPO has been shown to possess the same general biochemical properties of bDASPO, except that the former contains only FAD, while the latter is a mixture of two forms, one active containing FAD and one inactive containing 6-OH-FAD (9-20% depending on the preparation). This results in a slightly higher specific activity (about 15%) for rDASPO compared to bDASPO and in facilitated procedures for apoprotein preparation and reconstitution. Redox potentials of -97 mV and -157 mV were determined for free and l-(+)-tartrate complexed DASPO, respectively, in 0.1 M KPi, pH 7.0, 25 degrees C. The large positive shift in the redox potential of the coenzyme compared to free FAD (-207 mV) is in agreement with similar results obtained with other flavooxidases. rDASPO has been used to assess a possible oxidative activity of the enzyme towards a number of compounds used as agonists or antagonists of neurotransmitters, including d-aspartatic acid, d-glutamic acid, N-methyl-d-aspartic acid, d,l-cysteic acid, d-homocysteic acid, d, l-2-amino-3-phosphonopropanoic acid, d-alpha-aminoadipic acid, d-aspartic acid-beta-hydroxamate, glycyl-d-aspartic acid and cis-2, 3-piperidine dicarboxylic acid. Kinetic parameters for each substrate in 50 mM KPi, pH 7.4, 25 degrees C are reported.     

Chemical modification of functional arginyl residues in beef kidney D-aspartate oxidase.

Chemical modification of beef kidney D-aspartate oxidase by phenylglyoxal is a biphasic process involving the transient formation of an enzymatic species with a decreased activity versus dicarboxylic substrates, an increased activity versus D-proline and a new activity versus other monocarboxylic D-amino acids which is absent in the native protein. Prolonged incubation with the modifier causes complete inactivation of the enzyme. The presence of the competitive inhibitor L-tartrate in the incubation mixture prevents enzyme inactivation. Kinetic and structural data suggest that complete loss of activity is paralleled by modification of eight arginine residues, of which two are critical for the specificity and the activity of the enzyme. We propose that the two essential arginine residues are located in the substrate binding site of D-aspartate oxidase.     

Evaluation of the components of the insulin-like growth factors system in GH-deficient adults: effects of twelve-month rhGH treatment.

The impact of GH deficiency and rhGH replacement therapy on IGF-I, IGFBP-3 and ALS levels has been widely studied. There is less information available on IGF-II levels, the component of the ternary complex poorly dependent on GH. We investigate the components of IGFs system in 36 GHD adults (28M, 8F, age 45 +/- 14 yrs) before and after 12 months of rhGH therapy (mean dose 0.3 +/- 0.1 mg/day). One-hundred healthy sex- and age-matched subjects were studied for comparison. At baseline, GHD patients showed IGF-I and IGF-II levels and IGFs to IGFBP-3 molar ratios that were lower than controls. During therapy, IGF-I levels increased (p < 0.01) to normal range. IGF-II levels, though higher than at baseline (p < 0.01), remained lower than in controls (p < 0.01). ALS and IGFBP-3 significantly increased (p < 0.001). These modifications resulted in normalization in IGF-I to IGFBP-3 ratio, while no change in IGF-II to IGFBP-3 ratio was observed. In conclusion, the increase of serum IGF-II levels during rhGH treatment in GHD patients probably reflects the increase in the other components of ternary complex (ALS and IGFBP-3). However, serum IGF-II levels as well as IGF-II to IGFBP-3 ratio, although increased, were definitely lower than in controls. This last result, given the increasing evidences of a direct implication of IGF-II in cancer, may further confirm the safety of rhGH replacement in adults with severe GHD as diagnosed by appropriate stimulation tests.