Expression and purification of a Tuber borchii fruitbody-specific protein, TBF-1, from Escherichia coli: generation of polyclonal antibodies

TBF-1 is a fruitbody-specific protein present in the white truffle species Tuber borchii Vittad. A similar protein has been found only in the closely related species Tuber dryophilum (TDF-1), but not in other truffles. The protein from T. borchii was overexpressed as fusion protein in E. coli and was purified to homogeneity by affinity chromatography. Recombinant protein was used for generating polyclonal antibodies. The antiserum strongly reacted with TBF-1, weakly recognized TDF-1, and did not detect correlate band in the other white truffle species. The high level of expression of this protein in the fruitbody and the specificity of the antibody anti-TBF-1 make it possible to set up a diagnostic tool for detecting these species in natural samples and foodstuffs.     

Plasticity of glutamatergic control of striatal acetylcholine release in experimental parkinsonism: opposite changes at group-II metabotropic and NMDA receptors

To investigate whether adaptive changes of glutamatergic transmission underlie dysfunction of the cholinergic system in experimental parkinsonism, the effects of group-II metabotropic glutamate and NMDA receptor ligands on acetylcholine release was studied in striatal slices and synaptosomes obtained from naive rats, 6-hydroxydopamine hemi-lesioned rats and 6-hydroxydopamine hemi-lesioned rats chronically treated with levodopa (L-DOPA) plus benserazide (non-dyskinetic). Group-II metabotropic glutamate receptor agonists LY354740, DCG-IV and L-CCG-I inhibited the electrically-evoked endogenous acetylcholine release from slices, while NMDA facilitated it. LY354740 also inhibited K+-evoked acetylcholine release from synaptosomes. LY354740-induced inhibition was prevented by the group-II metabotropic glutamate receptor antagonist LY341495. In hemi-parkinsonian rats, sensitivity towards LY354740 was reduced while that to NMDA was enhanced in the lesioned (denervated) compared with unlesioned striatum. Moreover, dizocilpine inhibited acetylcholine release in the lesioned compared with unlesioned striatum. Chronic treatment with L-DOPA normalized sensitivity towards glutamatergic agonists. We conclude that striatal dopamine denervation results in plastic changes at group-II metabotropic glutamate and NMDA receptors that may shift glutamatergic control of acetylcholine release towards facilitation. From a clinical perspective, L-DOPA and NMDA antagonists appear effective in counteracting overactivity of striatal cholinergic interneurones associated with Parkinson's disease.     

The overexpressed hexahistidine-tagged human hexokinase type III is inhibited by D-glucose

Inhibition by its product, glucose, is a kinetic property of hexokinase type III. In this paper, we report the overexpression in Escherichia coli of human hexokinase type III. The recombinant enzyme was genetically fused with a hexahistidine peptide at the C-terminal end. This modification confers to the product the ability to bind the Ni2+ ion immobilised into agarose by nitrilotriacetic acid (NTA) groups. The purification was performed by one-step column chromatography using ammonium sulphate as stabilising agent. Recombinant hexokinase type III appears as a single band of approximately 100 kDa on a SDS-PAGE gel and shows specific activity of 16 U/mg. Its kinetic parameters are comparable to those of the native enzyme, including the fact that it can be inhibited by glucose. The comparison of these results with the properties of the overexpressed carboxyl-domain led us to suppose that the inhibition site for glucose required the presence of the N-terminal domain.     

Rabbit red blood cell hexokinase. Decay mechanism during reticulocyte maturation

In rabbit reticulocytes, the hexokinase (EC 2.7.1.1)-specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by a polyclonal antibody made in vitro shows that this maturation-dependent hexokinase decay is not due to accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system derived from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinae activity and the degradation of 125I-labeled enzyme. This degradation is ATP-dependent and requires both ubiquitin and a proteolytic fraction retained by DEAE-cellulose. Maximum ATP-dependent degradation was obtained at pH 7.5 in the presence of MgATP. MgGTP could replace MgATP with a relative stimulation of 0.90. 125I-Hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates that reach a steady-state concentration in 1 h, whereas the degradation of the enzyme is linear up to 8 h, suggesting that the formation of protein aggregates precedes enzyme catabolism. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mercaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugated to ubiquitin was shown by the formation of 125I-ubiquitin-hexokinase complexes in the presence of ATP and the enzymes of the ubiquitin-protein ligase system. Thus, the decay of hexokinase during reticulocyte maturation is ATP- and ubiquitin-dependent and suggests a new physiological role for the energy-dependent degradation system of reticulocytes.     

Chilling followed by incubation at 37 degrees C causes a reduction in NAD+ levels which can be prevented by the poly(ADP-ribose)transferase inhibitor 3-aminobenzamide

The exposure of cells for 60 min to a serum free medium at ice temperature followed by a return to normal culture conditions (30 min at 37 degrees C) caused a dramatic decrease in NAD+ levels. This decrease in NAD+ was prevented by 3-aminobenzamide. Alkaline elution analysis of DNA from cultures that were sisters to the ones utilized for measuring cellular NAD+ content revealed an absence of DNA breakage. These data suggest that poly(ADP-ribose)transferase may be induced in conditions not involving DNA fragmentation. The induction of this enzyme could therefore represent a cellular emergency reaction and not just a response to DNA damage.     

Bdnf expression in rat skeletal muscle after acute or repeated exercise

Brain derived growth factor (BDNF) gene of rat has a complex structure: at least four 5' untranslated exons regulated by different promoters and one 3' exon containing the encoding region. BDNF is expressed by skeletal muscles in an activity-dependent manner. In this study, BDNF mRNA was analysed by RT-PCR in the soleus muscle following a single (acute) session of running or a training of five days of running (repetitive exercise). Moreover, the expression of the exons was quantitatively analysed by real time RT-PCR. Finally, muscle BDNF protein level was evaluated by western blotting. BDNF mRNA was found to increase over the second day after acute exercise; on the other hand, two peaks (2 and 24 hours after the last session, respectively) in BDNF mRNA level were found after repetitive exercise, but it was similar to that of controls 6 hours after the last session. BDNF protein level progressively increased also after the mRNA went back to the basal level, so suggesting that it cumulates within the cell after acute exercise, whereas it followed the mRNA level time course after repetitive exercise. These results point to the following conclusions: BDNF mRNA is up-regulated by activity, but this response is delayed to the second day after acute exercise; repetitive exercise transiently depresses the expression of BDNF mRNA, so that the over-expression due to the previous day's exercise completely disappears 6 hours after the last exercise session.     

Restriction fragment length polymorphism species-specific patterns in the identification of white truffles

A molecular method for the identification of ectomycorrhizae belonging to five species of white truffle is described. The polymerase chain reaction (PCR) and universal primers were used to amplify internal transcribed spacers and 5.8S rDNA, target sequences present in a high number of copies. The amplified products were digested with restriction enzymes in order to detect interspecific polymorphisms. Species-specific restriction fragment length polymorphism patterns were determined for all five species. The use of PCR in conjunction with restriction enzymes provides a sensitive and efficient tool for use in distinguishing ectomycorrhizal species and monitoring inoculated seedlings or field mycorrhizal populations.     

Human erythrocyte hexokinases are immunologically related

Human erythrocytes contain three major hexokinase isoenzymes eluted by DE-52 chromatography between hexokinase type I and type II. Cross-reactivities of these isoenzymes were studied by means of a monospecific rabbit antibody against purified human placenta hexokinase type I. It was shown that the three hexokinase isoenzymes were immunologically related, supporting the concept of a postsynthetic mechanism(s) as their origin.