Cooperative effect of inositol hexakisphosphate, bezafibrate, and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin

The cooperative effect of inositol hexakisphosphate (IHP), bezafibrate (BZF), and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of IHP, BZF, and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of 2,3-diphosphoglycerate (2,3-DPG), which have been attributed to a low affinity conformation of the tetramer. Addition to HbNO of two allosteric effectors together (such as IHP and BZF, or IHP and CFA) further stabilizes the low affinity conformation of the ligated hemoprotein (i.e., HbNO). Moreover, in the presence of saturating amounts of IHP, the affinity of BZF and CFA for HbNO increases by about fifteenfold. Likewise, in the presence of both IHP and BZF, as well as in IHP and CFA, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced with respect to that observed in the presence of IHP, BZF, or CFA alone, which in turn is lower than that reported in the absence of any allosteric effector. All the data were obtained at pH 7.0 (in 1.0 × 10⁻¹ M N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]/NaOH buffer system plus 1.0 × 10⁻¹ M NaCl), as well as at 100 K and/or 20°C. The results here reported represent clearcut evidence for the cooperative and specific (i.e., functionally relevant) binding of IHP, BZF, and CFA to Hb.     

Cytosolic 5′-nucleotidase/nucleoside phosphotransferase: A nucleoside analog activating enzyme?

Nucleoside phosphotransferase acting on inosine and deoxyinosine has been partially purified from cultured Chinese hamster lung fibroblasts (V79). The activity is associated with a cytosolic 5′-nucleotidase acting on IMP and deoxyIMP. The transfer of the phosphate group from IMP to inosine catalyzed by this enzyme was activated by ATP and 2,3-bisphosphoglycerate. Inosine, deoxyinosine, guanosine, deoxyguanosine, and the nucleoside analogs 2′,3′-dideoxyinosine and 8-azaguanosine are substrates, while adenosine and deoxyadenosine are not. IMP, deoxyIMP, GMP, and deoxyGMP are the best phosphate donors. The cytosolic 5′-nucleotidase/phosphotransferase substrate, 8-azaguanosine, was found to be very toxic for cultured fibroblasts (LD₅₀ = 0.32 μM). Mutants resistant to either 8-azaguanosine and the correspondent base 8-azaguanine were isolated and characterized. Our results indicated that the 8-azaguanosine-resistant cells were lacking both cytosolic 5′-nucleotidase and hypoxanthine-guanine phosphoribosyltransferase, while 8-azaguanine resistant cells were lacking only the latter enzyme. Despite this observation, both mutants displayed 8-azaguanosine resistance, thus indicating that cytosolic 5′-nucleotidase is not essential for the activation of this nucleoside analog.     

Synergistic effect of phosphatidylserine with γ-aminobutyric acid in antagonizing the isoniazid-induced convulsions in mice

The influence of phosphatidylserine (PS) on the isoniazid-induced convulsions has been studied in mice. Sonicated dispersions of this phospholipid given intravenously do not show anticonvulsant activity but they do so when -aminobutyric acid (GABA) is simultaneously injected. GABA alone is inactive. The synergism between PS and GABA is influenced by the structure of the phospholipid liposomes. In contrast to multilamellar vesicles, oligolamellar vesicles are active. Under these conditions the effect shows head group specificity, in that the neutral phosphatidylcholine (PC) or the acidic phosphatidylinositol (PI) are inactive, either in the presence or in the absence of GABA. Lysophosphatidylserine (lysoPS), the deacylated PS derivative, shows increased efficacy as an isoniazid antagonist in the presence of GABA, and has anticonvulsant activity also in the absence of GABA. Other lysophospholipids are inactive. It is suggested that PS, after its metabolic conversion to lysoPS, enhances the anticonvulsant effect of GABA.     

How Skill Expertise Shapes the Brain Functional Architecture: An fMRI Study of Visuo-Spatial and Motor Processing in Professional Racing-Car and Na?ve Drivers

The present study was designed to investigate the brain functional architecture that subserves visuo-spatial and motor processing in highly skilled individuals. By using functional magnetic resonance imaging (fMRI), we measured brain activity while eleven Formula racing-car drivers and eleven ‘naïve’ volunteers performed a motor reaction and a visuo-spatial task. Tasks were set at a relatively low level of difficulty such to ensure a similar performance in the two groups and thus avoid any potential confounding effects on brain activity due to discrepancies in task execution. The brain functional organization was analyzed in terms of regional brain response, inter-regional interactions and blood oxygen level dependent (BOLD) signal variability. While performance levels were equal in the two groups, as compared to naïve drivers, professional drivers showed a smaller volume recruitment of task-related regions, stronger connections among task-related areas, and an increased information integration as reflected by a higher signal temporal variability. In conclusion, our results demonstrate that, as compared to naïve subjects, the brain functional architecture sustaining visuo-motor processing in professional racing-car drivers, trained to perform at the highest levels under extremely demanding conditions, undergoes both ‘quantitative’ and ‘qualitative’ modifications that are evident even when the brain is engaged in relatively simple, non-demanding tasks. These results provide novel evidence in favor of an increased ‘neural efficiency’ in the brain of highly skilled individuals.     

Bone morphogenetic protein-9 activates Smad and ERK pathways and supports human osteoclast function and survival in vitro

BMP-9 is a potent osteogenic factor; however, its effects on osteoclasts, the bone-resorbing cells, remain unknown. To determine the effects of BMP-9 on osteoclast formation, activity and survival, we used human cord blood monocytes as osteoclast precursors that form multinucleated osteoclasts in the presence of RANKL and M-CSF in long-term cultures. BMP-9 did not affect osteoclast formation, but adding BMP-9 at the end of the culture period significantly increased bone resorption compared to untreated cultures, and reduced both the rate of apoptosis and caspase-9 activity. BMP-9 also significantly downregulated the expression of pro-apoptotic Bid, but only after RANKL and M-CSF, which are both osteoclast survival factors, had been eliminated from the culture medium. To investigate the mechanisms involved in the effects of BMP-9, we first showed that osteoclasts expressed some BMP receptors, including BMPR-IA, BMPR-IB, ALK1, and BMPR-II. We also found that BMP-9 was able to induce the phosphorylation of Smad-1/5/8 and ERK 1/2 proteins, but did not induce p38 phosphorylation. Finally, knocking down the BMPR-II receptor abrogated the BMP-9-induced ERK-signaling, as well as the increase in bone resorption. In conclusion, these results show for the first time that BMP-9 directly affects human osteoclasts, enhancing bone resorption and protecting osteoclasts against apoptosis. BMP-9 signaling in human osteoclasts involves the canonical Smad-1/5/8 pathway, and the ERK pathway.     

Conformation-sensitive antibodies against alzheimer amyloid-beta by immunization with a thioredoxin-constrained B-cell epitope peptide

Immunotherapy against the amyloid-beta (Abeta) peptide is a valuable potential treatment for Alzheimer disease (AD). An ideal antigen should be soluble and nontoxic, avoid the C-terminally located T-cell epitope of Abeta, and yet be capable of eliciting antibodies that recognize Abeta fibrils and neurotoxic Abeta oligomers but not the physiological monomeric species of Abeta. We have described here the construction and immunological characterization of a recombinant antigen with these features obtained by tandem multimerization of the immunodominant B-cell epitope peptide Abeta1-15 (Abeta15) within the active site loop of bacterial thioredoxin (Trx). Chimeric Trx(Abeta15)n polypeptides bearing one, four, or eight copies of Abeta15 were constructed and injected into mice in combination with alum, an adjuvant approved for human use. All three polypeptides were found to be immunogenic, yet eliciting antibodies with distinct recognition specificities. The anti-Trx(Abeta15)4 antibody, in particular, recognized Abeta42 fibrils and oligomers but not monomers and exhibited the same kind of conformational selectivity against transthyretin, an amyloidogenic protein unrelated in sequence to Abeta. We have also demonstrated that anti-Trx(Abeta15)4, which binds to human AD plaques, markedly reduces Abeta pathology in transgenic AD mice. The data indicate that a conformational epitope shared by oligomers and fibrils can be mimicked by a thioredoxin-constrained Abeta fragment repeat and identify Trx(Abeta15)4 as a promising new tool for AD immunotherapy.     

Proteome analysis of whole saliva: a new tool for rheumatic diseases--the example of Sjögren's syndrome

Sj?gren's syndrome (pSS) is a systemic disease that affects salivary glands directly, and is therefore expected to influence the composition of human whole saliva (WS) fluid. The aim of this study was to characterize the WS proteins of pSS patients using a proteomic approach to assess a valid procedure to examine the global changes of the salivary protein profiles in connective tissue disorders. The WS proteins expressed in patients affected by pSS and healthy volunteers were analyzed using the 2-DE technique. The WS protein pattern was altered in pSS patients compared to controls, with a decrease in some of the typical salivary proteins. Particularly, a remarkable alteration of carbonic anhydrase VI was observed. Moreover, a comparison of WS protein profile of pSS patients with the one obtained from controls revealed a set of differentially expressed proteins. These proteins were related to acute and chronic inflammation while some others were involved in oxidative stress injury. These findings are in line with the systemic immuno-inflammatory aspects of pSS and open the possibility for a systematic search of diagnostic biomarkers and targets for therapeutic intervention in pSS.     

Role of yeasts in the qualitative structuring of extra virgin olive oil

This review sought to describe the role played by some components of the microbiota of extra virgin olive oil (EVOO), particularly yeasts, in structuring the physicochemical and sensorial quality of freshly produced olive oil. Yeasts can survive during the entire storage period of the product. To date, approximately 25 yeast species isolated from oil produced in more than six countries have been identified, eight of which are classified as new species. Some yeast species improve the health qualities of oil, whereas many others improve the chemical composition and sensory characteristics based on β-glucosidase and esterase enzymes, which are involved in the hydrolysis of the bitter glucoside known as oleuropein. However, some species, which are typically favoured by the high water content in the oily matrix, such as lipase-producing yeasts, can worsen the initial chemical characteristics of EVOO oil during storage. Some physical treatments that are compatible with the EVOO production specification affect the biotic component of the oil by reducing the concentration of yeasts. The possibility of minimizing the invasive action on the biotic component of the oil by appropriately selecting the physical treatment for each oil is discussed.     

Studies of mercurated derivatives of acetylacetone and ethyl acetoacetate by solid state nuclear magnetic resonance and vibrational spectroscopies

Seven new mono- and/or dimercurated compounds involving acetylacetone (2,4-pentanedione) or ethyl acetoacetate (3-ethyl ketobutanoate) were synthesized in aqueous medium. In all cases, mercuration occurred at methylene carbon atoms. All compounds were carefully analyzed by solid state carbon-13 nuclear magnetic resonance. Assignments were confirmed by using selective sequences, which allowed a total editing of the spectra. It was shown that deshielding of ¹³C mercurated sites occurred when the rate of mercuration increased. It was also possible to measure direct ¹J(¹⁹⁹Hg¹³C) coupling constants. The main bands of the vibrational spectra (infrared and Raman) were assigned. It was proved that v(C = O) and δ(C(γ)-H) could be directly related to the mercuration rate of molecules.