Contralateral input modulates the excitability of dorsal horn neurons involved in noxious signal processes. Potential role in neuronal sensitization

Wide Dynamic Range (WDR) neurons in the spinal cord receive inputs from the contralateral side that, under normal conditions, are ineffective in generating an active response. These inputs are effective when the target WDRs change their excitability conditions. To further reveal the mechanisms supporting this effectiveness shift, we investigated the weight of the excitation of the contralateral neurons on the target WDR responses. In the circuit of presynaptic (sending) and postsynaptic (receiving) neurons in crossed spinal connections the fibres that form the presynaptic neurons impinge on postsynaptic neurons can be considered the final relay of this contralateral pathway. The enhancement of the presynaptic neuron excitability may thus modify the efficacy of the contralateral input. Pairs of neurons each on a side of the spinal cord, at the L5-L6 lumbar level were simultaneously recorded in intact, anaesthetized, paralysed rats. The excitatory aminoacid NMDA and strychnine, the antagonist of the inhibitory aminoacid glycine, were iontophoretically administrated to presynaptic neurons to increase their excitability. Before and during the drug administration, spontaneous and noxious-evoked activities of the neurons were analysed. During the iontophoresis of the two substances we found that noxious stimuli applied to the receptive field of presynaptic neurons activated up to 50% of the previously unresponsive postsynaptic neurons on the opposite side. Furthermore, the neurons on both sides of the spinal cord showed significantly increased spontaneous activity and amplified responses to ipsilateral noxious stimulation. These findings indicate that the contralateral input participates in the circuit dynamics of spinal nociceptive transmission, by modulating the excitability of the postsynaptic neurons. A possible functional role of such a nociceptive transmission circuit in neuronal sensitization following unilateral nerve injury is hypothesized.     

Genetic characterization of Listeria monocytogenes food isolates and pathogenic potential within serovars 1/2a and 1/2b

A total of 39 Listeria monocytogenes strains isolated from raw milk, smoked meat, chicken carcass and reference strains, belonging to serovars 1/2a, 4a, 1/2b, 3b and 4b, were analysed by RAPD and by polymorphisms of the virulent genes inlAB and iap. Ten isolates, belonging to serovars 1/2a and 1/2b and, collected from raw milk and smoked meat, were further tested for pathogenicity by IP injection into mice. The clustering of the 39 L. monocytogenes strains in 3 groups at 0.45 similarity level, based on molecular typing, was observed. Distribution of serovars in these clusters was in agreement with the proposed three Listeria monocytogenes lineages. Within serovar 1/2b, the 50% lethal dose (LD50) ranged from 8.4 x 10(4) to 1.7 x 10(6) cfu.ml(-1). One of the serovar 1/2b strains, isolated from smoked meat, exhibited the lowest virulence potential evaluated by LD50 and by mean time to death (MTD) and, from this point of view, was completely different from the other strains. Our results suggest the existence of heterogeneity in virulence levels within serovars 1/2a and 1/2b. However, when comparing the isolates based on genotyping, virulence indicators and food origin, no relation could be assessed.     

Mutations of the T-cell receptor constant region after in utero stem cell transplantation

Like the immunoglobulin genes, the T-cell receptor genes are generated by rearrangements of non-contiguous genomic V, D and J regions, but unlike the immunoglobulin genes, somatic hypermutation is an infrequent event in T-cell receptor genes. Here, we describe the occurrence of spontaneous mutations in the constant regions of the T-cell receptor beta chains of T lymphocytes obtained from two babies who underwent in utero transplantation because of severe combined immunodeficiency. In view of the fact that in babies receiving transplants before birth, hematopoietic chimerism is consistently present, the lymphocytes are likely to be under chronic activation, which may represent a relevant biologic stimulus for generating the observed T-cell receptor hypermutation. This possibility is supported by the finding that the highest number of mutations was identified in clonally expanded T cells. These results provide further support indicating that hypermutation of the T-cell receptor genes may indeed occur, given the necessary conditions.     

Monitoring the early steps of unfolding of dicalcium and mono-Ce3+-substituted forms of P43M calbindin D9k

Early steps of unfolding of P43M Calbindin D(9k) have been evaluated by NMR spectroscopy on the native dicalcium and on the paramagnetic monocerium-substituted derivative. Although at 2 M GdmHCl the protein core maintains its overall folding and structure, amide (15)N R(2) measurements and cross correlation rates between N-H dipole-dipole relaxation and (15)N CSA relaxation reveal a closer and stronger packing of the hydrophobic interactions in the protein as a response to the presence of denaturing agents in solution. A complete reorientation of the Met43 side chain toward the hydrophobic core is accomplished by the disappearance of the millisecond dynamics observed on the native form of Calbindin D(9k), while cross correlation rates provide evidence that the two-way hydrogen bond between Leu23 and Val61 is broken or substantially weakened. The substitution of the calcium ion in site II with the paramagnetic Ce(3+) ion allowed us to obtain a number of long-range nonconventional constraints, namely, pseudocontact shifts, which were used, together with the NOEs collected on the native state, to monitor subtle structural variations occurring in the non-native state of the protein. Although the average rmsd between the structures of native and non-native states is small (0.48 A), structural rearrangements could be reliably identified. Our results provide unprecedented information about the behavior of Calbindin D(9k) during the early steps of unfolding. Furthermore, they constitute strong evidence of the efficiency of paramagnetism-based constraints in monitoring subtle structural changes that are beyond the sensitivity of an approach based only on NOE.     

Mutations in the met oncogene unveil a "dual switch" mechanism controlling tyrosine kinase activity

The met oncogene, encoding the high affinity hepatocyte growth factor receptor, is the only known gene inherited in human cancer that is invariably associated with somatic duplication of the mutant locus. Intriguingly, mutated Met requires ligand stimulation in order to unleash its transforming potential. Furthermore, individuals bearing a germ line met mutation develop cancer only late in life and with incomplete penetrance. To date, there is no molecular explanation for this unique behavior, which is unusual for a dominant oncogene. Here we investigate the molecular mechanisms underlying met oncogenic conversion by generating antibodies specific for the differently phosphorylated forms of the Met protein. Using these antibodies, we show that activation of wild-type Met is achieved through sequential phosphorylation of Tyr1235 and Tyr1234 in the activation loop and that mutagenesis of either tyrosine dramatically impairs kinase function. Surprisingly, oncogenic Met mutants never become phosphorylated on Tyr1234 despite their high enzymatic activity, and mutagenesis of Tyr1234 does not affect their biochemical or biological function. By analyzing the enzymatic properties of the mutant proteins in different conditions, we demonstrate that oncogenic mutations do not elicit constitutive kinase activation but simply overcome the requirement for the second phosphorylation step, thus reducing the threshold for activation. In the presence of activating signals, these mutations result therefore in a dynamic imbalance toward the active conformation of the kinase. This explains why mutant met provides an oncogenic predisposition but needs a second activating "hit," provided by sustained ligand stimulation or receptor overexpression, to achieve a fully transformed phenotype.     

The tailspike protein of Shigella phage Sf6. A structural homolog of Salmonella phage P22 tailspike protein without sequence similarity in the beta-helix domain

Bacteriophage Sf6 tailspike protein is functionally equivalent to the well characterized tailspike of Salmonella phage P22, mediating attachment of the viral particle to host cell-surface polysaccharide. However, there is significant sequence similarity between the two 70-kDa polypeptides only in the N-terminal putative capsid-binding domains. The major, central part of P22 tailspike protein, which forms a parallel beta-helix and is responsible for saccharide binding and hydrolysis, lacks detectable sequence homology to the Sf6 protein. After recombinant expression in Escherichia coli as a soluble protein, the Sf6 protein was purified to homogeneity. As shown by circular dichroism and Fourier transform infrared spectroscopy, the secondary structure contents of Sf6 and P22 tailspike proteins are very similar. Both tailspikes are thermostable homotrimers and resist denaturation by SDS at room temperature. The specific endorhamnosidase activities of Sf6 tailspike protein toward fluorescence-labeled dodeca-, deca-, and octasaccharide fragments of Shigella O-antigen suggest a similar active site topology of both proteins. Upon deletion of the N-terminal putative capsid-binding domain, the protein still forms a thermostable, SDS-resistant trimer that has been crystallized. The observations strongly suggest that the tailspike of phage Sf6 is a trimeric parallel beta-helix protein with high structural similarity to its functional homolog from phage P22.     

Comparative in vitro study on the long term performance of cemented hip stems: validation of a protocol to discriminate between "good" and "bad" designs

The long-term clinical success of cemented hip stems is influenced both by the implant design, and by the surgical procedure. A methodology is proposed for discriminating between implant designs with different clinical outcomes. The protocol was designed with industrial pre-clinical validation in mind.Two cemented stem types were tested, one (Lubinus SPII) having good and the other (Müller Curved) having poor clinical outcomes. Three implants for each type were subjected to a mechanical in vitro test of one million loading cycles. Each cycle reproduced the load components of stair climbing. Interface shear micromotion was measured during the test in the direction of rotation and along the stem axis. The stem roughness before and after the test was compared. After the test, the cement mantles were retrieved and inspected through dye penetrants to detect evidences of micro-damage. For each specimen, the events of the loosening process were examined, based on the in vitro data available, so as to analyze the whole failure mechanism.The protocol developed was sensitive to the implant design, with significantly different results being found for the two stem types, both in terms of stem-cement micromotions, surface roughness alteration, and cement mantle damage. The information yielded by the three different investigation techniques was consistent for each of the two groups of specimens tested, allowing a better understanding of the failure process. In vitro inducible micromotion and permanent migration measurements, together with cement-stem interface fretting damage and cement fatigue damage, can help predicting the clinical performance of cemented stems.     

Identification of a mutated receptor-like protein tyrosine phosphatase kappa as a novel,class II HLA-restricted melanoma antigen

Recent studies increasingly point to a pivotal role of CD4(+) T cells in human anti-tumor immune response. Here we show that lymphocytes purified from a tumor-infiltrated lymph node of a melanoma patient that had remained disease free for 10 years after surgical resection of a lymph node metastasis comprised oligoclonal class II HLA-restricted CD4(+) T cells recognizing the autologous tumor cells in vitro. In fact, the CD4(+) T cell clones isolated from these lymphocytes displayed a tumor-specific, cytotoxic activity in addition to a Th1-like cytokine profile. By a genetic approach, a peptide derived from a mutated receptor-like protein tyrosine phosphatase kappa was identified as a novel HLA-DR10-restricted epitope for all the melanoma-specific CD4(+) T cell clones. The immunogenic peptide was shown to contain the mutated residue that was crucial for T cell recognition and activation. Moreover, a systemic immunity against the mutated peptide was detectable in the patient's peripheral blood T lymphocytes obtained during the disease-free period of follow-up. These findings further support the relevance of CD4(+) T cells directed against mutated epitopes in tumor immunity and provide the rationale for a possible usage of mutated, tumor-specific Ags for immunotherapy of human cancer.     

Synthesis and pharmacological characterization of functionalized 2-pyridones structurally related to the cardiotonic agent milrinone

A new class of cardiotonic agents characterized by a 2-pyridone structure was synthesized. Appropriate sym-2-dimethylaminomethylene-1,3-diones reacted with methylcyanoacetate to afford the desired compounds. These derivatives were evaluated for their ability in inducing cardiotonic response on guinea pig isolated myocardial preparations. Compound 8b increased atrial contractility to an extent which is significantly higher than that of milrinone, the parent drug used as a reference compound. The pharmacological characterization and the docking studies performed on 8b highlighted its selective mechanism of action via type 3 PDE (PDE3) inhibition.     

Mitochondrial production of oxygen radical species and the role of Coenzyme Q as an antioxidant

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), which is considered as the pathogenic agent of many diseases and of aging. We have investigated the role of complex I in superoxide radical production and found by the combined use of specific inhibitors of complex I that the one-electron donor to oxygen in the complex is a redox center located prior to the sites where three different types of Coenzyme Q (CoQ) competitors bind, to be identified with an Fe-S cluster, most probably N2, or possibly an ubisemiquinone intermediate insensitive to all the above inhibitors. Short-chain Coenzyme Q analogs enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analog, idebenone, is particularly effective, raising doubts on its safety as a drug. Cells counteract oxidative stress by antioxidants. CoQ is the only lipophilic antioxidant to be biosynthesized. Exogenous CoQ, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases. The plasma membrane oxidoreductase and DT-diaphorase are two such systems, likewise, they are overexpressed under oxidative stress conditions.