Kinetics of post-exercise phosphate transport in human skeletal muscle: an in vivo 31P-MR spectroscopy study

31-Phosphorus magnetic resonance spectroscopy was used to investigate in vivo the kinetics of inorganic phosphate transport and intracellular pH after exercise in human skeletal muscle. Intracellular pH further decreased from the value reached at the end of work showing a minimum between 25 and 45 sec and then increased back to the resting value. Inorganic phosphate showed an initial fast rate of recovery corresponding to the decreasing phase of pH, and a second phase in which a slow rate of recovery corresponded to increasing pH. The biphasic patterns of both phosphate and pH recoveries are in agreement with and support in vitro evidence that Pi transport into mitochondria is modulated by pH.     

Self/nonself recognition in Tuber melanosporum is not mediated by a heterokaryon incompatibility system

Vegetative incompatibility is a widespread phenomenon in filamentous ascomycetes, which limits formation of viable heterokaryons. Whether this phenomenon plays a role in maintaining the homokaryotic state of the hyphae during the vegetative growth of Tuber spp. Gene expression, polymorphism analysis as well as targeted in vitro experiments allowed us to test whether a heterokaryon incompatibility (HI) system operates in Tuber melanosporum. HI is controlled by different genetic systems, often involving HET domain genes and their partners whose interaction can trigger a cell death reaction. Putative homologues to HI-related genes previously characterized in Neurospora crassa and Podospora anserina were identified in the T. melanosporum genome. However, only two HET domain genes were found. In many other ascomycetes HET domains have been found within different genes including some members of the NWD (NACHT and WD-repeat associated domains) gene family of P. anserina. More than 50 NWD homologues were found in T. melanosporum but none of these contain a HET domain. All these T. melanosporum paralogs showed a conserved gene organization similar to the microexon genes only recently characterized in Schistosoma mansoni. Expression data of the annotated HI-like genes along with low allelic polymorphism suggest that they have cellular functions unrelated to HI. Moreover, morphological analyses did not provide evidence for HI reactions between pairs of genetically different T. melanosporum strains. Thus, the maintenance of the genetic integrity during the vegetative growth of this species likely depends on mechanisms that act before hyphal fusion.     

Balanced biochemical reactions: a new approach to unify chemical and biochemical thermodynamics

A novel procedure is presented which, by balancing elements and electric charge of biochemical reactions which occur at constant pH and pMg, allows assessing the thermodynamics properties of reaction Δ_rG ^′0, Δ_rH ^′0, Δ_rS ^′0 and the change in binding of hydrogen and magnesium ions of these reactions. This procedure of general applicability avoids the complex calculations required by the use of the Legendre transformed thermodynamic properties of formation Δ_fG ^′0, Δ_fH ^′0 and Δ_fS ^′0 hitherto considered an obligatory prerequisite to deal with the thermodynamics of biochemical reactions. As a consequence, the term “conditional” is proposed in substitution of “Legendre transformed” to indicate these thermodynamics properties. It is also shown that the thermodynamic potential G is fully adequate to give a criterion of spontaneous chemical change for all biochemical reactions and then that the use of the Legendre transformed G′ is unnecessary. The procedure proposed can be applied to any biochemical reaction, making possible to re-unify the two worlds of chemical and biochemical thermodynamics, which so far have been treated separately.     

Bacteria-produced ferric exopolysaccharide nanoparticles as iron delivery system for truffles (Tuber borchii)

Applied Microbiology and Biotechnology - Iron exopolysaccharide nanoparticles were biogenerated during ferric citrate fermentation by Klebsiella oxytoca DSM 29614. Before investigating their...     

First evidence for truffle production from plants inoculated with mycelial pure cultures

Truffle (Tuber spp.) cultivation is based on raising mycorrhizal trees in greenhouses that have been inoculated with suspensions of ascospores. The problem with this is that pests, pathogens, and other mycorrhizal fungi can contaminate the trees. Furthermore, because ascospores are produced sexually, each plant potentially has a different genetic mycorrhizal makeup from each other so tailoring the mycorrhizal component of plants to suit a particular set of soil and climatic conditions is out of the question. Here, we report on the production of Tuber borchii-mycorrhized plants using pure cultures, establishing a truffière with these and subsequent production of its fruiting bodies. This study opens up the possibility of producing commercial numbers of Tuber-mycorrhized trees for truffle cultivation using mycelial inoculation techniques. It also poses questions about the mechanism of fertilization between the different strains which were located in different parts of the experimental truffière.     

The role of pH on the thermodynamics and kinetics of muscle biochemistry: an in vivo study by (31)P-MRS in patients with myo-phosphorylase deficiency

In this study we assessed ΔG'(ATP) hydrolysis, cytosolic [ADP], and the rate of phosphocreatine recovery using Phosphorus Magnetic Resonance Spectroscopy in the calf muscle of a group of patients affected by glycogen myo-phosphorylase deficiency (McArdle disease). The goal was to ascertain whether and to what extent the deficit of the glycogenolytic pathway would affect the muscle energy balance. A typical feature of this pathology is the lack of intracellular acidosis. Therefore we posed the question of whether, in the absence of pH decrease, the rate of phosphocreatine recovery depends on the amount of phosphocreatine consumed during exercise. Results showed that at the end of exercise both [ADP] and ΔG'(ATP) of patients were significantly higher than those of matched control groups reaching comparable levels of phosphocreatine concentration. Furthermore, in these patients we found that the rate of phosphocreatine recovery is not influenced by the amount of phosphocreatine consumed during exercise. These outcomes provide experimental evidence that: i) the intracellular acidification occurring in exercising skeletal muscle is a protective factor for the energy consumption; and ii) the influence of pH on the phosphocreatine recovery rate is at least in part related to the kinetic mechanisms of mitochondrial creatine kinase enzyme.     

Characterization of Tuber borchii and Arbutus unedo mycorrhizas

For the first time, arbutoid mycorrhizas established between Tuber borchii and Arbutus unedo were described. Analyzed mycorrhizas were from one T. borchii natural truffle ground, dominated by Pinus pinea, as well as synthesized in greenhouse conditions. A. unedo mycorrhizas presented some typical characteristics of ectomycorrhizas of T. borchii. However, as in arbutoid mycorrhizas, ramification was cruciform and intracellular colonization in epidermal cells was present. The ability of T. borchii to form ectomycorrhizas with A. unedo opens up the possibility to also use this fruit plant for truffle cultivation. This represents an important economic opportunity in Mediterranean areas by combining both the cultivation of precious truffles and the production of edible fruits which are used fresh or in food delicacies.     

The homeodomain transcription factor Prep1 (pKnox1) is required for hematopoietic stem and progenitor cell activity

Most of the hypomorphic Prep1^i/i embryos (expressing 3-10% of the Prep1 protein), die between E17.5 and P0, with profound anemia, eye malformations and angiogenic anomalies [Ferretti, E., Villaescusa, J.C., Di Rosa, P., Fernandez-Diaz, L.-C., Longobardi, E., Mazzieri, R., Miccio, A., Micali, N., Selleri, L., Ferrari G., Blasi, F. (2006). Hypomorphic mutation of the TALE gene Prep1 (pKnox1) causes a major reduction of Pbx and Meis proteins and a pleiotropic embryonic phenotype. Mol. Cell. Biol. 26, 5650-5662]. We now report on the hematopoietic phenotype of these embryos. Prep1^i/i fetal livers (FL) are hypoplastic, produce less common myeloid progenitors colonies (CFU-GEMM) in cytokine-supplemented methylcellulose and have an increased number of B-cells precursors that differentiate poorly. Prep1^i/i FL is able to protect lethally irradiated mice only at high cell doses but the few protected mice show major anomalies in all hematopoietic lineages in both bone marrow (BM) and peripheral organs. Prep1^i/i FL cells compete inefficiently with wild type bone marrow in competitive repopulation experiments, suggesting that the major defect lies in long-term repopulating hematopoietic stem cells (LTR-HSC). Indeed, wt embryonic expression of Prep1 in the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), cKit⁺Sca1⁺Lin⁻AA4.1⁺ (KSLA) cells and B-lymphocytes precursors agrees with the observed phenotype. We therefore conclude that Prep1 is required for a correct and complete hematopoiesis.     

Interactions of nucleic acids with distamycins. Binding of Dst-3 to d(CGTTTAAACG)2 and d(CGTACGTACG)2.

The binding between Distamycin 3 and the palindromic duplexes d(CGTTTAAACG)2 and d(CGTACGTACG)2 was investigated by two independent techniques: UV-Vis absorption in the Job's plot approach and Induced Circular Dichroism. Both decamers bind two molecules of peptide per duplex, with close overall affinities. This result indicates that a row of six A:T base pairs can accommodate two molecules of drug and that the minimal binding site of Distamycin 3 can consist of just two A:T base pairs.     

In vivo assessment of human skeletal muscle mitochondria respiration in health and disease

One of many problems to be faced when assessing in vivo human muscle mitochondria respiration by phosphorus magnetic resonance spectroscopy (³¹P-MRS) is the definition of the correct reference population and the values of reference range. To take into account most factors that influence muscle activity as age, sex, physical activity; nutritional state etc., an exceedingly high number of different reference groups are needed. To overcome this problem we developed specific tests to assess separately in vivo the activity and the functionality of muscle mitochondria by ³¹P-MRS in clinical settings. By activity we refer to muscle whole metabolic activity, i.e. the total oxidative capacity of muscle mitochondria which is influenced by many factors (age, sex, physical activity, nutritional state etc.). By functionality we refer to the qualitative aspects of mitochondrial respiration which depends on the integrity of mitochondrial multienzyme systems and on substrate availability. Our tests ha ve been experienced on some 1200 patients and are currently used to detect deficits of mitochondrial respiration and ion transport in patients with suspected primary or secondary muscle mitochondrial malfunctioning. (Mol Cell Biochem 174: 11–15, 1997)