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.     

Dynein-related polypeptides in pollen and pollen tubes

 Microtubules in pollen tubes are evident within the vegetative and generative cell cytoplasm. This observation led to the formulation of several hypotheses regarding the role of microtubules in cytoplasmic movement and the migration of the vegetative nucleus/generative cell along the pollen tube. The study of microtubular motor proteins in pollen tubes followed the discovery and characterization of an immunoreactive homolog of mammalian kinesin in tobacco pollen tubes. Recent identification of dynein-related polypeptides in pollen tubes of Nicotiana tabacum and pollen of Ginkgo biloba is a significant step in the definition of the role of microtubule function within pollen and pollen tubes. Received: 31 May 1996 / Revision accepted: 26 July 1996     

Influence of glycerol on the structure and redox properties of horse heart cytochrome c. A circular dichroism and electrochemical study

The effect of glycerol on the structure and redox properties of horse heart cytochrome c was investigated by absorption spectroscopy, circular dichroism, and dc cyclic voltammetry techniques. The results show that the organic solvent increases the -helix structure of the protein and induces slight changes at the active-site environment; however, the overall tertiary structure does not appear to be significantly perturbed. Glycerol stabilizes cytochrome c, the free energy of denaturation (G ₀) being approximately 0.7 kcal/mol larger than that determined in phosphate buffer under the same conditions, and influences the heterogeneous electron transfer kinetics at a chemically modified gold electrode; on the other hand, the redox potential of the protein is unaltered. On the whole, the results obtained indicate that glycerol acts as a suitable stabilizing agent of cytochrome c, which is of interest for application in biotechnology; the organic solvent does not alter the tertiary structure significantly or the redox properties of the protein. This has to be interpreted not only in terms of the glycerol-induced solvent ordering around the protein surface, but also as due to the specific features of the protein matrix.     

Intramolecular photoredox reactions in iron(III) cytochrome c and its azide derivative

The irradiation of deaerated solutions of horse heart cytochrome c causes the reduction of Fe(III) to Fe(II). The dependence of the photoreaction quantum yield on pH shows that the photoreactive species is a form of cytochrome c which contains methionine-80 and histidine-18 as heme ligands. The primary photochemical event consists of an electron transfer from the sulphur of methionine- 80 to iron. The re-oxidation of the photochemically obtained Fe(II) protein gives a Fe(III) cytochrome which exhibits a typical low-spin absorption spectrum, lacking the 695-nm band and indicating that a strong field ligand, other than methionine-80, coordinates to the sixth binding site of the heme iron. Spectrophotometric titration of the photochemically modified Fe(III) cytochrome shows that histidine- 18 remains bound in the fifth position.The substitution of methionine-80 with the more oxidizable azide ligand increases the efficiency of the intramolecular electron transfer. Azide radicals, detected by spin-trapping ESR technique, are formed in the primary act. Visible-UV spectral data indicate that histidine-18 and methionine-80 occupy the fifth and sixth position, respectively, in the photoreaction product. All the results obtained correlate well with those previously obtained in investigations concerning the photoredox behavior of iron porphyrin complexes.