Amino acids and protein profile in floral nectar: Much more than a simple reward

Nectar is considered a primary alimentary reward for a large variety of pollinators. Recent studies demonstrated that nectar may have other functions in addition to attracting pollinators. Mainly other two functions have been recognized: defense against microbial invasion and post-secretion modification of sugar profile. The floral nectar of Cucurbita pepo L. has been analyzed to confirm the presence of defense proteins, namely xylosidases that were identified by means of a proteomic approach in a previous study. An enzyme assay was also performed that revealed low invertase activity for which the optimal pH was determined. This invertase activity is not able to modify the sugar profile significantly during the short period of anthesis (6 h). The amino acids complement of the nectar of both sexes was also determined. Sixteen of the 20 protein amino acids have been detected. Proline comprises more than 30% of total amino acid content in male and female flowers. Three non-protein amino acids (taurine, β-alanine, and GABA) represent almost one third of the total amino acid content, and of these, GABA is the most abundant (16%). Several biological activities were attributed to these amino acids and further studies are needed to assess their presumed involvement in managing the foraging behavior of insects. More recent studies, including our own, demonstrate that the various functions of nectar are much more complex than previously thought.     

Conversion of cytochrome c into a peroxidase: inhibitory mechanisms and implication for neurodegenerative diseases

A further function of cytochrome c (cyt c), beyond respiration, is realized outside mitochondria in the apoptotic program. In the early events of apoptosis, the interaction of cyt c with a mitochondrion-specific phospholipid, cardiolipin (CL), brings about a conformational transition of the protein and acquirement of peroxidase activity. The hallmark of cyt c with peroxidase activity is its partial unfolding accompanied by loosening of the Fe sixth axial bond and an enhanced access of the heme catalytic site to small molecules like H2O2. To investigate the peroxidase activity of non-native cyt c, different forms of the protein were analyzed with the aim to correlate their structural features with the acquired enzymatic activity and apoptogenic properties (wt cyt c/CL complex and two single cyt c variants, H26Y and Y67H, free and bound to CL). The results suggest that cyt c may respond to different environments by changing its fold thus favouring the exertion of different biological functions in different pathophysiological cell conditions. Transitions among different conformations are regulated by endogenous molecules such as ATP and may be affected by synthetic molecules such as minocycline, thus suggesting a mechanism explaining its use as therapeutic agent impacting on disease-associated oxidative and apoptotic mechanisms.     

Involvement of adenosine A1 and A2A receptors in (-)-linalool-induced antinociception

In recent studies performed in our laboratory we have shown that acute administration of (-)-linalool, the natural occurring enantiomer in essential oils, possesses anti-inflammatory, antihyperalgesic and antinociceptive effects in different animal models. The antihyperalgesic and antinociceptive effects of (-)-linalool have been ascribed to its capacity in stimulating the opioidergic, cholinergic and dopaminergic systems, as well as to its interaction with K+ channels, or to its local anaesthetic activity and/or to the negative modulation of glutamate transmission. Activation of A1 or A2A receptors has been shown to induce antinociceptive effects, and the possible involvement of adenosine in (-)-linalool antinociceptive effect, has not been elucidated yet. Therefore, in the present study, we have investigated the effects of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective adenosine A1 receptor antagonist and the effects of 3,7-dimethyl-1-propargilxanthine (DMPX), a selective adenosine A2A receptor antagonist on the antinociception of (-)-linalool in mice, measured in the hot-plate test. Both DPCPX (0.1 mg/kg; i.p.) and DMPX (0.1 mg/kg; i.p.) pre-treatment significantly depressed the antinociceptive effect of (-)-linalool at the highest doses tested. These findings demonstrated that the effect of (-)-linalool on pain responses is, at least partially, mediated by the activity of adenosine A1 and A2A receptors.     

Ypt32p and Mlc1p bind within the vesicle binding region of the class V myosin Myo2p globular tail domain

Myosin V is an actin-based motor essential for a variety of cellular processes including skin pigmentation, cell separation and synaptic transmission. Myosin V transports organelles, vesicles and mRNA by binding, directly or indirectly, to cargo-bound receptors via its C-terminal globular tail domain (GTD). We have used the budding yeast myosin V Myo2p to shed light on the mechanism of how Myo2p interacts with post-Golgi carriers. We show that the Rab/Ypt protein Ypt32p, which associates with membranes of the trans -Golgi network, secretory vesicles and endosomes and is related to the mammalian Rab11, interacts with the Myo2p GTD within a region previously identified as the 'vesicle binding region'. Furthermore, we show that the essential myosin light chain 1 (Mlc1p), required for vesicle delivery at the mother-bud neck during cytokinesis, binds to the Myo2p GTD in a region overlapping that of Ypt32p. Our data are consistent with a role of Ypt32p and Mlc1p in regulating the interaction of post-Golgi carriers with Myo2p subdomain II.     

Salivary cortisol concentration after high-intensity interval exercise: Time of day and chronotype effect

Due to personal and working necessities, the time for exercise is often short, and scheduled early in the morning or late in the afternoon. Cortisol plays a central role in the physiological and behavioral response to a physical challenge and can be considered as an index of exercise stress. Therefore, the aim of this study was to evaluate the influence of the circadian phenotype classification on salivary cortisol concentration in relation to an acute session of high-intensity interval exercise (HIIE) performed at different times of the day. Based on the morningness–eveningness questionnaire, 12 M-types (N = 12; age 21 ± 2 years; height 179 ± 5 cm; body mass 74 ± 12 kg, weekly training volume 8 ± 1 hours) and 11 E-types (N = 11; age 21 ± 2 years; height 181 ± 11 cm; body mass 76 ± 11 kg, weekly training volume 7 ± 2 hours) were enrolled in a randomized crossover study. All subjects underwent measurements of salivary cortisol secretion before (PRE), immediately after (POST), and 15 min (+15 min), 30 min (+30 min), 45 min (+45 min) and 60 min (+60 min) after the completion of both morning (08.00 am) and evening (08.00 p.m.) high-intensity interval exercise. Two-way analysis of variance with Tuckey’s multiple comparisons test showed significant increments over PRE-cortisol concentrations in POSTcondition both in the morning (4.88 ± 1.19 ng · mL^?1 vs 6.60 ± 1.86 ng · mL^?1, +26.1%, P < 0.0001, d > 0.8) and in the evening (1.56 ± 0.48 ng · mL^?1 vs 2.34 ± 0.37, +33.4%, P = 0.034, d > 0.6) exercise in all the 23 subject that performed the morning and the evening HIIE. In addition, during morning exercise, significant differences in cortisol concentration between M-types and E-types at POST (5.49 ± 0.98 ng · mL^?1 versus 8.44 ± 1.08 ng · mL^?1, +35%, P < 0.0001, d > 0.8), +15 min (4.52 ± 0.42 ng · mL^?1 versus 6.61 ± 0.62 ng · mL^?1, +31.6%, P < 0.0001, d > 0.8), +30 min (4.10 ± 1.44 ng · mL^?1 versus 6.21 ± 1.60 ng · mL^?1, +34.0%, P < 0.0001, d = 0.7), + 45 min (3.78 ± 0.55 ng · mL^?1 versus 5.80 ± 0.72 ng · mL^?1, +34.9%, P < 0.0001, d = 0.7), and + 60 min condition(3.53 ± 0.45 ng · mL^?1 versus 5.78 ± 1.13 ng · mL^?1, 38.9%, P = 0.0008, d = 0.7) were noted. No statistical significant differences between M-types and E-types during evening HIIE on post-exercise cortisol concentration were detected. E-types showed a higher morning peak of salivary cortisol respect to M-types when performing a HIIE early in the morning and produced higher salivary cortisol concentrations after the cessation of the exercise. Practical applications suggest that it is increasingly important for the exercise professionals to identify the compatibility between time of day for exercising and chronotype to find the individual’s favorable circadian time to perform a HIIE.     

Anion- and pH-linked effects on the heme-iron geometry in ferrous nitrosylated monomeric myoglobins

 The cooperative effect of anions and proton concentration on the EPR spectroscopic properties of the ferrous nitrosylated derivative of monomeric Mb from loggerhead sea turtle (Caretta caretta), sperm whale (Physeter catodon), and horse (Caballus caballus) has been investigated between pH 4.5 and 9.0, at 100 K. In the absence of anions, an EPR spectrum characteristic of the hexa-coordinated species of ferrous nitrosylated Mb with an axial geometry is observed, which is unaffected by pH. On the other hand, a transition toward a species characterized by an EPR spectrum corresponding to a hexa-coordinated rhombic geometry takes place in the presence of phosphate, acetate, citrate, sulfate, and chloride. Only the hexa-coordinated form characterized by the rhombic EPR spectrum appears then to undergo a pH-dependent transition toward the penta-coordinated species. Present results show clear-cut evidence for the spectroscopic coupling of proton and anion binding sites with the Mb reactive center, indicating that an allosteric mechanism might modulate the proximal HisF8-heme-NO geometry in monomeric hemoproteins. Received: 15 December 1997 / Accepted: 15 June 1998     

Different structural effects of allosteric modulators on subunits of tetrameric ferrous nitrosylated human hemoglobin: an EPR spectroscopic study

 The X-band EPR spectroscopic features of the ferrous nitrosylated derivative of α(Fe)₂β(Co)₂ and of α(Co)₂β(Fe)₂ metal hybrids of human hemoglobin (Hb) have been investigated at pH 7.0 and analyzed in parallel with those of the native nitrosylated tetramer (HbNO). The effect of 2,3-biphosphoglycerate (BPG), inositol hexakisphosphate (IHP) and bezafibrate (BZF) has been investigated in order to understand the perturbations induced on α and β subunits in the tetramer by the binding of allosteric effectors. A large perturbation is observed in both subunits upon BZF binding, while in the case of IHP only α-chains are affected; on the other hand, BPG leaves both chains essentially unperturbed. Thus, different binding modes of allosteric effectors to HbNO may occur, and the simultaneous addition of two effector molecules, namely BPG and BZF or IHP and BZF to HbNO, brings about different alterations of the X-band EPR spectroscopic properties. This behavior indicates that the intramolecular communication pathway(s) between the heme and the binding pockets of the heterotropic ligands (i.e., IHP and BZF, or BPG and BZF) are different, leading to distinct structural perturbations. Received: 19 September 1997 / Accepted: 16 December 1997     

Inosine stimulates chemotaxis, Ca2+-transients and actin polymerization in immature human dendritic cells via a pertussis toxin-sensitive mechanism independent of adenosine receptors

Inosine is an endogenous purine nucleoside, which is formed by adenosine deaminidase during adenosine breakdown and is released into the extracellular space from the sympathetic nervous system or injured cells. Here, we studied the biological activity of inosine on human dendritic cells (DC), which are specialized antigen presenting cells characterized by their ability to migrate from the blood to peripheral tissues, and then to secondary lymphoid organs where they initiate adaptive immune responses. In immature DC, inosine concentration-dependently stimulated Ca(2+)-transients, actin polymerization, and chemotaxis. Experiments with adenosine receptor antagonists and pertussis toxin (PTX) as well as desensitization studies suggested that the activity of inosine was mediated by a G protein-coupled receptor pathway independent of adenosine receptors. DC, induced to mature by lipopolysaccharide, lost their ability to respond towards inosine with these activities. Moreover, inosine did neither influence membrane expression of CD54, CD80, CD83, CD86, HLA-DR, and MHC class I molecules nor modulated secretion of interleukin (IL)-12, IL-10, and tumor necrosis factor alpha in immature and lipopolysaccharide-matured DC. In aggregate, our study indicates that inosine may be involved in the trafficking control system of immature DC, and mediates its chemotactic activity by a PTX-sensitive mechanism independent of adenosine receptors.     

Anti-HIV properties of cationic fullerene derivatives

A series of regioisomeric bis-fulleropyrrolidines bearing two ammonium groups have been synthesized and their activities against HIV-1 and HIV-2 have been evaluated. Two trans isomers have been endowed with interesting antiviral properties, confirming the importance of the relative positions of the substituent on the C(60) cage. In addition, reduced amphiphilicity of molecules to other compounds previously reported decreases their cytotoxicity in CEM cell cultures. None of the compounds showed any inhibitory activity against a variety of DNA and RNA viruses other than HIV.