Abnormal nociception and opiate sensitivity of STOP null mice exhibiting elevated levels of the endogenous alkaloid morphine

Calcitonin gene-related peptide (CGRP) plays an important role in peripheral and central sensitization. CGRP also is a key molecule in the spino-parabrachial-amygdaloid pain pathway. Blockade of CGRP1 receptors in the spinal cord or in the amygdala has antinociceptive effects in different pain models. Here we studied the electrophysiological mechanisms of behavioral effects of CGRP in the amygdala in normal animals without tissue injury. Whole-cell patch-clamp recordings of neurons in the latero-capsular division of the central nucleus of the amygdala (CeLC) in rat brain slices showed that CGRP (100 nM) increased excitatory postsynaptic currents (EPSCs) at the parabrachio-amygdaloid (PB-CeLC) synapse, the exclusive source of CGRP in the amygdala. Consistent with a postsynaptic mechanism of action, CGRP increased amplitude, but not frequency, of miniature EPSCs and did not affect paired-pulse facilitation. CGRP also increased neuronal excitability. CGRP-induced synaptic facilitation was reversed by an NMDA receptor antagonist (AP5, 50 μM) or a PKA inhibitor (KT5720, 1 μM), but not by a PKC inhibitor (GF109203X, 1 μM). Stereotaxic administration of CGRP (10 μM, concentration in microdialysis probe) into the CeLC by microdialysis in awake rats increased audible and ultrasonic vocalizations and decreased hindlimb withdrawal thresholds. Behavioral effects of CGRP were largely blocked by KT5720 (100 μM) but not by GF109203X (100 μM). The results show that CGRP in the amygdala exacerbates nocifensive and affective behavioral responses in normal animals through PKA- and NMDA receptor-dependent postsynaptic facilitation. Thus, increased CGRP levels in the amygdala might trigger pain in the absence of tissue injury.     

Ultrastructure of long-range transport carriers moving from the trans Golgi network to peripheral endosomes

The delivery of mannose 6-phosphate receptors carrying lysosomal hydrolases from the trans-Golgi network (TGN) to the endosomal system is mediated by selective incorporation of the receptor-hydrolase complexes into vesicular transport carriers (TCs) that are coated with clathrin and the adaptor proteins, GGA and AP-1. Previous electron microscopy (EM) and biochemical studies have shown that these TCs consist of spherical coated vesicles with a diameter of 60-100 nm. The use of fluorescent live cell imaging, however, has revealed that at least some of this transport relies on a subset of apparently larger and highly pleiomorphic carriers that detach from the TGN and translocate toward the peripheral cytoplasm until they meet with distally located endosomes. The ultrastructure of such long-range TCs has remained obscure because of the inability to examine by conventional EM the morphological details of rapidly moving organelles. The recent development of correlative light-EM has now allowed us to obtain ultrastructural 'snapshots' of these TCs immediately after their formation from the TGN in live cells. This approach has revealed that such carriers range from typical 60- to 100-nm clathrin-coated vesicles to larger, convoluted tubular-vesicular structures displaying several coated buds. We propose that this subset of TCs serve as vehicles for long-range distribution of biosynthetic or recycling cargo from the TGN to the peripheral endosomes.     

Structural roles of the active site iron(III) ions in catechol 1,2-dioxygenases and differential secondary structure changes in isoenzymes A and B from Acinetobacter radioresistens S13

The reversible active site metal ion removal process for two catechol 1,2-dioxygenase isoenzymes (IsoA and IsoB) isolated from Acinetobacter radioresistens S13 has been monitored using circular dichroism and fluorescence spectroscopic techniques. IsoA and IsoB are homodimers, containing one iron(III) ion per subunit. Their amino acid sequence identity is 48.4%. Previous experiments suggested that structural diversities could be responsible for the differential thermal and pH stabilities of the two isoenzymes and of their distinct demetallation kinetics. The far-UV CD spectra of IsoA and IsoB catechol 1,2-dioxygenases from A. radioresistens S13 provide information on their secondary structures. IsoB appears to have a content of alpha-helices higher than IsoA. Upon metal ion removal, both proteins reversibly lose part of their secondary structure following distinct pathways. CD spectra simulations allowed us to estimate the content of alpha-helices, beta-sheets, and turns for each isoenzyme and to monitor the secondary structure rearrangements. The metal ion withdrawal has large influence on the secondary structure: in particular a significant reduction of alpha-helices content is observed for both isoenzymes. Intrinsic fluorescence emission spectra clearly support such results, adding information on the local environment changes of the tryptophan residues. The positioning of Trp250 in IsoB has been shown to be of particular interest for monitoring the local structure changes occurring upon metal ion removal. For the first time these studies allow to underline the role of active site iron ions on dioxygenases folding and stability, further evidencing the differences in structural assembling between the two isoenzymes from A. radioresistens S13.     

NMR-based metabonomic study of transgenic maize

The aim of this research was to verify the possibility of identifying and classifying maize seeds obtained from transgenic plants, in different classes according to the modification, on the basis of the concerted variation in metabolite levels detected by NMR spectra. It was possible to recognise the discriminant metabolites of transgenic samples as well as to classify non-a priori defined samples of maize. It is important to underline that the obtained results are useful to point out the metabolic consequences of a specific genic modification on a plant, without using a targeted analysis of the different metabolites, in fact it was possible to classify the seeds also without the complete assignment of the spectra. The analysis was performed by applying multivariate techniques (principal component analysis and partial least squares-discriminant analysis) to NMR data.     

Declusterization of GABAA receptors affects the kinetic properties of GABAergic currents in cultured hippocampal neurons

Speed and reliability of synaptic transmission are essential for information coding in neuronal networks and require the presence of clustered neurotransmitter receptors at the plasma membrane in precise apposition to presynaptic terminals. Receptor clusterization is the result of highly regulated processes involving functional and structural proteins. Among the structural elements, microtubules are known to play a crucial role in anchoring of gamma-aminobutyric acid, type A (GABA(A)) receptors. Here we show that microtubule depolymerization with nocodazole induces the declusterization of GABA(A) receptors and modifies the kinetic properties of GABAergic currents in cultured hippocampal neurons. In particular, this drug, applied either in the bath or via the patch pipette, induced the acceleration of the onset kinetics of miniature inhibitory postsynaptic currents (mIPSCs) without significantly affecting their frequency, thus suggesting a main postsynaptic site of action. After nocodazole treatment, current responses to ultrafast applications of GABA exhibited a faster rise time and an accelerated onset of desensitization. A quantitative analysis of GABA-evoked currents and model simulations suggest that declusterization affects the gating properties of GABA(A) receptors. In particular, a faster entry into the desensitized state of declustered GABA(A) receptors may account for the changes in the kinetic properties of mIPSCs after nocodazole treatment. Hence it appears that the clustered condition of GABA(A) receptors contributes in shaping GABAergic currents.     

Study of high molecular weight wheat glutenin subunit 1Dx5 by 13C and 1H solid-state NMR spectroscopy. I. Role of covalent crosslinking

This work describes a carbon and proton solid-state NMR study of the hydration of a high molecular weight wheat glutenin subunit, 1Dx5. The effect of the presence of disulfide bonds on the hydration behavior of the subunit is investigated by a comparison of the unalkylated and alkylated forms of the protein. Hydration induces partial plasticization of the protein so that some segments become more mobile than others. The 13C cross-polarization and magic-angle spinning (MAS) spectra of the samples in the dry state and at two hydration levels (approximately 40 and approximately 65% D2O) were used to monitor the protein fraction resisting plasticization (trains). Conversely, 13C single pulse excitation and 1H-MAS experiments were used to gain information on the more plasticized segments (loops). The molecular motion of the two protein dynamic populations was further characterized by 13C T1 and 1H T(1rho), T2, and T1 relaxation times. The results suggest that hydration leads to the formation of a network held by a cooperative action of hydrogen bonded glutamines and some hydrophobic interactions. The looser protein segments are suggested to be glycine- and glutamine-rich segments. The primary structure is therefore expected to significantly determine the proportion of trains and loops in the network. The presence of disulfide bonds was observed to promote easier plasticization of the protein and the formation of a more mobile network, probably involving a higher number of loops and/or larger loops.     

p66SHC promotes apoptosis and antagonizes mitogenic signaling in T cells

Of the three Shc isoforms, p66Shc is responsible for fine-tuning p52/p46Shc signaling to Ras and has been implicated in apoptotic responses to oxidative stress. Here we show that human peripheral blood lymphocytes and mouse thymocytes and splenic T cells acquire the capacity to express p66Shc in response to apoptogenic stimulation. Using a panel of T-cell transfectants and p66Shc(-/-) T cells, we show that p66Shc expression results in increased susceptibility to apoptogenic stimuli, which depends on Ser36 phosphorylation and correlates with an altered balance in apoptosis-regulating gene expression. Furthermore, p66Shc blunts mitogenic responses to T-cell receptor engagement, at least in part by transdominant inhibition of p52Shc signaling to Ras/mitogen-activated protein kinases, in an S36-dependent manner. The data highlight a novel interplay between p66Shc and p52Shc in the control of T-cell fate.     

Dietary habits and plasma fatty acids levels in a population of Italian children: is there any relationship?

The relationships between dietary habits and the blood fatty acid (FA) profile have been poorly explored, particularly in children. Aim of the present investigation was to look at the associations between dietary habits and the plasma pattern of FA in a school-age population. In 105 healthy 8-years old children, the nutritional habits have been evaluated by means of a Food Frequency Questionnaire and a 24-h recall. FA analyses (expressed as FA%) were performed by means of capillary gas chromatography. Statistics: non-parametric tests. The children with high pasta and low red meat consumption (n = 9), compared to those with either medium or low pasta and either high or medium red meat consumption (n = 96), showed lower levels of total saturated (29% vs. 35%, P = 0.001) and higher levels of total monounsaturated (30% vs. 24%, P = 0.04) FA in plasma. They also showed lower levels of LDL cholesterol (90 vs. 109 mg/dl, P = 0.08) and fasting insulin (4.0 vs. 6.3 microU/ml, P = 0.04) in blood. A high consumption of pasta coupled with a low intake of red meat may be marker of a food behaviour and/or lifestyle associated with a more favourable pattern of circulating FA and hematochemical metabolic markers.