Biological time-dependent difference in effect of peroxynitrite demonstrated by the mouse hot plate pain model

We previously demonstrated the rhythmic pattern of L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) cascade in nociceptive processes. The coupled production of excess NO and superoxide leads to the formation of an unstable intermediate peroxynitrite, which is primarily responsible for NO-mediated toxicity. In the present study, we evaluated the biological time-dependent effects of exogenously administered peroxynitrite on nociceptive processes and peroxynitrite-induced changes in the analgesic effect of morphine using the mouse hot-plate pain model. Experiments were performed at four different times of day (1, 7, 13, and 19 hours after lights on, i.e., HALO) in mice of both sexes synchronized to a 12 h:12 h light-dark cycle. Animals were injected intraperitoneally (i.p.) with saline or 10 mg/kg morphine 30 min before and 0.001 mg/kg peroxynitrite 30 sec before hot-plate testing, respectively. The analgesic effect of morphine exhibited significant biological time-dependent differences in the thermally-induced algesia; whereas, administration of peroxynitrite alone exhibited either significant algesic or analgesic effect, depending on the circadian time of its injection. Concomitant administration of peroxynitrite and morphine reduced morphine-induced analgesia at three of the four different study time points. In conclusion, peroxynitrite displayed nociceptive and antinociceptive when administered alone according to the circadian time of treatment, while it diminished analgesic activity when administered in combination with morphine at certain biological times.     

Vanillin bioproduction from alkaline hydrolyzate of corn cob by Escherichia coli JM109/pBB1

Hydrolysis of corn cob performed for 6 h with 0.5 N NaOH at solid/liquid ratio of 0.084 g/g allowed obtaining a hydrolyzate containing 1171 ± 34 mg/l ferulic acid and 2156 ± 63 mg/l p-coumaric acid that was used as a medium for vanillin bioproduction by the engineered strain Escherichia coli JM109/pBB1. Aiming at maximizing vanillin bioproduction, the effects of medium heat sterilization, one-stage or two-stage pre-cultivation, adaptation of the microorganism to the hydrolyzate and inoculum biomass level were investigated. Biomass pre-cultivated once in unsterilized hydrolyzate was able to effectively convert ferulic and p-coumaric acids to a mixture of vanillin, vanillic acid and vanillyl alcohol provided with the typical vanilla flavor. At initial biomass concentration of 0.5 g_DM/l, maximum values of vanillin concentration (239 ± 15 mg/l), vanillin yield on consumed ferulic acid (0.66 ± 0.03 mol/mol) and vanillin volumetric productivity (10.9 ± 0.7 mg/lh) were obtained after 22 h.     

Contribution of Herpesvirus Specific CD8 T Cells to Anti-Viral T Cell Response in Humans

Herpesviruses infect most humans. Their infections can be associated with pathological conditions and significant changes in T cell repertoire but evidences of symbiotic effects of herpesvirus latency have never been demonstrated. We tested the hypothesis that HCMV and EBV-specific CD8 T cells contribute to the heterologous anti-viral immune response. Volume of activated/proliferating virus-specific and total CD8 T cells was evaluated in 50 patients with acute viral infections: 20 with HBV, 12 with Dengue, 12 with Influenza, 3 with Adenovirus infection and 3 with fevers of unknown etiology. Virus-specific (EBV, HCMV, Influenza) pentamer+ and total CD8 T cells were analyzed for activation (CD38/HLA-DR), proliferation (Ki-67/Bcl-2_low) and cytokine production. We observed that all acute viral infections trigger an expansion of activated/proliferating CD8 T cells, which differs in size depending on the infection but is invariably inflated by CD8 T cells specific for persistent herpesviruses (HCMV/EBV). CD8 T cells specific for other non-related non persistent viral infection (i.e. Influenza) were not activated. IL-15, which is produced during acute viral infections, is the likely contributing mechanism driving the selective activation of herpesvirus specific CD8 T cells. In addition we were able to show that herpesvirus specific CD8 T cells displayed an increased ability to produce the anti-viral cytokine interferon-γ during the acute phase of heterologous viral infection. Taken together, these data demonstrated that activated herpesvirus specific CD8 T cells inflate the activated/proliferating CD8 T cells population present during acute viral infections in human and can contribute to the heterologous anti-viral T cell response.     

A new interpretation of P300 responses upon analysis of coherences

Previous studies on cognitive dynamics showed that oscillatory responses of P300 are composed of mainly delta and theta responses. In the present study, for the first time, the long-distance intra-hemispheric event related coherence (auditory oddball paradigm) and evoked coherence (simple sound) were compared in order to evaluate the effects of cognitive tasks on the long-distance coherences. Seventeen healthy subjects (8 female, 9 male) were included in the study. The coherence was analyzed for delta (1–3.5 Hz), theta (4–7.5 Hz) and alpha (8–13 Hz) frequency ranges for (F₃-P₃, F₄-P₄, F₃-T₇, F₄-T₈, F₃-O_1, F₄-O₂) electrode pairs. The coherence to target responses were higher than the non-target and simple auditory response coherence. This difference is significant for the delta coherence for both hemispheres and for theta coherences over the left hemisphere. The highest coherences were recorded at fronto-temporal locations for all frequency bands (delta, theta, alpha). Furthermore, fronto-parietal coherences were higher than the fronto-occipital coherences for all frequency bands (delta, theta, alpha).These results show that the fronto-temporal and fronto-parietal connections are most relevant for the identification of the target signal. This analysis open the way for a new interpretation of dynamic localization results during cognitive tasks.     

Structure-based analysis of protein dynamics: comparison of theoretical results for hen lysozyme with X-ray diffraction and NMR relaxation data

An analytical approach based on Gaussian network model (GNM) is proposed for predicting the rotational dynamics of proteins. The method, previously shown to successfully reproduce X-ray crystallographic temperature factors for a series of proteins is extended here to predict bond torsional mobilities and reorientation of main chain amide groups probed by 15N-H nuclear magnetic resonance (NMR) relaxation. The dynamics of hen egg-white lysozyme (HEWL) in the folded state is investigated using the proposed approach. Excellent agreement is observed between theoretical results and experimental (X-ray diffraction and NMR relaxation) data. The analysis reveals the important role of coupled rotations, or cross-correlations between dihedral angle librations, in defining the relaxation mechanism on a local scale. The crystal and solution structures exhibit some differences in their local motions, but their global motions are identical. Hinge residues mediating the cooperative movements of the alpha- and beta-domains are identified, which comprise residues in helix C, Glu35 and Ser36 on the loop succeeding helix B, Ile55 and Leu56 at the turn between strands II and III. The central part of the beta-domain long loop and the turn between strands I and II display an enhanced mobility. Finally, kinetically hot residues and key interactions are identified, which point at helix B and beta-strand III as the structural elements underlying the stability of the tertiary structure.     

Fast-folding protein kinetics,hidden intermediates,and the sequential stabilization model

Do two‐state proteins fold by pathways or funnels? Native‐state hydrogen exchange experiments show discrete nonnative structures in equilibrium with the native state. These could be called hidden intermediates (HI) because their populations are small at equilibrium, and they are not detected in kinetic experiments. HIs have been invoked as disproof of funnel models, because funnel pictures appear to indicate (1) no specific sequences of events in folding; (2) a continuum, rather than a discrete ladder, of structures; and (3) smooth landscapes. In the present study, we solve the exact dynamics of a simple model. We find, instead, that the present microscopic model is indeed consistent with HIs and transition states, but such states occur in parallel, rather than along the single pathway predicted by the sequential stabilization model. At the microscopic level, we observe a huge multiplicity of trajectories. But at the macroscopic level, we observe two pathways of specific sequences of events that are relatively traditional except that they are in parallel, so there is not a single reaction coordinate. Using singular value decomposition, we show an accurate representation of the shapes of the model energy landscapes. They are highly complex funnels.     

Identification of kinetically hot residues in proteins.

A number of recent studies called attention to the presence of kinetically important residues underlying the formation and stabilization of folding nuclei in proteins, and to the possible existence of a correlation between conserved residues and those participating in the folding nuclei. Here, we use the Gaussian network model (GNM), which recently proved useful in describing the dynamic characteristics of proteins for identifying the kinetically hot residues in folded structures. These are the residues involved in the highest frequency fluctuations near the native state coordinates. Their high frequency is a manifestation of the steepness of the energy landscape near their native state positions. The theory is applied to a series of proteins whose kinetically important residues have been extensively explored: chymotrypsin inhibitor 2, cytochrome c, and related C2 proteins. Most of the residues previously pointed out to underlie the folding process of these proteins, and to be critically important for the stabilization of the tertiary fold, are correctly identified, indicating a correlation between the kinetic hot spots and the early forming structural elements in proteins. Additionally, a strong correlation between kinetically hot residues and loci of conserved residues is observed. Finally, residues that may be important for the stability of the tertiary structure of CheY are proposed.     

Temporal Variation in Serum Nitrite Levels in Rats and Mice

Although considerable evidence implicates involvement of nitric oxide (NO) in circadian regulation, little is known about possible 24h variations in basal NO metabolism. In this study, daily variations in serum nitrite levels were studied in locally bred mice and rats during the months of September and October. The serum was separated from blood samples obtained at six different times of the day and night (lh, 5h, 9h, 13h, 17h, and 21h after lights off [HALO] from male albino mice and rats). As an index of in vivo NO generation, serum nitrite levels (determined by the diazotization method) in rats exhibited significant temporal fluctuation (unpaired Student t test), with the concentration highest at 5 HALO and 21 HALO and lowest at 9 HALO. No such temporal variation was detected in mice in these studies conducted on locally bred animals in the autumn. (Chwnobiology International, 16(4), 527-532, 1999)     

Coarse-grained simulations of conformational dynamics of proteins: Application to apomyoglobin

A coarse-grained dynamic Monte Carlo method is proposed for investigating the conformational dynamics of proteins. Each residue is represented by two interaction sites, one at the α-carbon, and the other on the amino acid sidechain. Geometry and energy parameters extracted from databank structures are used. The method is applied to the crystal structure of apomyoglobin (apo-Mb). Equilibrium and dynamic properties of apo-Mb are characterized within computation times one order of magnitude shorter than conventional molecular dynamics (MD) simulations. The calculated rms fluctuations in α-carbons are in good agreement with crystallographic temperature factors. Regions exhibiting enhanced conformational mobilities are identified. Among the loops connecting the eight helices A to H, the loop CD undergoes the fastest motions, leading to partial unwinding of helix D. Helix G is the most stable helix on the basis of the kinetic stability of dihedral angles, followed by the respective helices A, E, H, and B. These results, in agreement with H/D exchange and two-dimensional NMR experiments, as well as with MD simulations, lend support to the use of the proposed approach as an efficient, yet physically plausible, means of characterizing protein conformational dynamics. Proteins 31:271–281, 1998. © 1998 Wiley-Liss, Inc.