Circulating gammadelta T cells in young/adult and old patients with cutaneous primary melanoma

BACKGROUND: In a previous study we demonstrated the existence of numerical and functional alterations of gammadelta T cells in healthy elderly. Recently, we analysed the involvement of gammadelta T lymphocytes in malignant melanoma, describing a lower frequency of circulating gammadelta T cells, an altered pattern of cytokine production, and an impaired in vitro expansion of these cells in primary cutaneous melanoma patients. METHODS: In this study we investigated the existence of numerical and functional alterations of circulating gammadelta T cells in young/adult and old melanoma patients, comparing the data obtained with age-matched healthy subjects. RESULTS: We demonstrated that the number of circulating gammadelta+ T cells was significantly and similarly reduced in young/adult and old melanoma patients and in old healthy subjects in comparison with young healthy donors. The decrease was due to a reduction of Vdelta2 T cells whereas the number of Vdelta1 T cells was not affected. A higher percentage of gammadelta+ T cells producing TNF-alpha was found in old healthy donors, whereas a reduced number of TNF-alpha-producing gammadelta+ T cells was present in melanoma patients independently by age. No significant difference was observed in IFN-gamma production. After a 10-day in vitro culture, both the percentage and the expansion index of gammadelta T cells, and in particular of Vdelta2 subset, were significantly and similarly reduced both in young/adult and old melanoma patients, and in healthy aged people, in comparison with young/adult healthy subjects. CONCLUSIONS: Our study demonstrates that the numerical and functional impairment of gammadelta T cells found in melanoma patients is not correlated with age and that it has characteristics very similar to the alterations of gammadelta T cells found in old healthy subjects. We suggest that a similar impairment of gammadelta T cell population may be related to the increased susceptibility to tumors present in the elderly as well as in the pathogenesis of malignant melanoma.     

In vivo and in vitro characterization of novel neuronal plasticity factors identified following spinal cord injury

Following spinal cord injury, there are numerous changes in gene expression that appear to contribute to either neurodegeneration or reparative processes. We utilized high density oligonucleotide microarrays to examine temporal gene profile changes after spinal cord injury in rats with the goal of identifying novel factors involved in neural plasticity. By comparing mRNA changes that were coordinately regulated over time with genes previously implicated in nerve regeneration or plasticity, we found a gene cluster whose members are involved in cell adhesion processes, synaptic plasticity, and/or cytoskeleton remodeling. This group, which included the small GTPase Rab13 and actin-binding protein Coronin 1b, showed significantly increased mRNA expression from 7-28 days after trauma. Overexpression in vitro using PC-12, neuroblastoma, and DRG neurons demonstrated that these genes enhance neurite outgrowth. Moreover, RNAi gene silencing for Coronin 1b or Rab13 in NGF-treated PC-12 cells markedly reduced neurite outgrowth. Coronin 1b and Rab13 proteins were expressed in cultured DRG neurons at the cortical cytoskeleton, and at growth cones along with the pro-plasticity/regeneration protein GAP-43. Finally, Coronin 1b and Rab13 were induced in the injured spinal cord, where they were also co-expressed with GAP-43 in neurons and axons. Modulation of these proteins may provide novel targets for facilitating restorative processes after spinal cord injury.     

Novel symmetric and asymmetric DNA scission determinants for Streptococcus pneumoniae topoisomerase IV and gyrase are clustered at the DNA breakage site

Topoisomerase (topo) IV and gyrase are bacterial type IIA DNA topoisomerases essential for DNA replication and chromosome segregation that act via a transient double-stranded DNA break involving a covalent enzyme-DNA "cleavage complex." Despite their mechanistic importance, the DNA breakage determinants are not understood for any bacterial type II enzyme. We investigated DNA cleavage by Streptococcus pneumoniae topo IV and gyrase stabilized by gemifloxacin and other antipneumococcal fluoroquinolones. Topo IV and gyrase induce distinct but overlapping repertoires of double-strand DNA breakage sites that were essentially identical for seven different quinolones and were augmented (in intensity) by positive or negative supercoiling. Sequence analysis of 180 topo IV and 126 gyrase sites promoted by gemifloxacin on pneumococcal DNA revealed the respective consensus sequences: G(G/c)(A/t)A*GNNCt(T/a)N(C/a) and GN4G(G/c)(A/c)G*GNNCtTN(C/a) (preferred bases are underlined; disfavored bases are in small capitals; N indicates no preference; and asterisk indicates DNA scission between -1 and +1 positions). Both enzymes show strong preferences for bases clustered symmetrically around the DNA scission site, i.e. +1G/+4C, -4G/+8C, and particularly the novel -2A/+6T, but with no preference at +2/+3 within the staggered 4-bp overhang. Asymmetric elements include -3G and several unfavored bases. These cleavage preferences, the first for Gram-positive type IIA topoisomerases, differ markedly from those reported for Escherichia coli topo IV (consensus (A/G)*T/A) and gyrase, which are based on fewer sites. However, both pneumococcal enzymes cleaved an E. coli gyrase site suggesting overlap in gyrase determinants. We propose a model for the cleavage complex of topo IV/gyrase that accommodates the unique -2A/+6T and other preferences.     

7-Nitro-2,1,3-benzoxadiazole derivatives, a new class of suicide inhibitors for glutathione S-transferases. Mechanism of action of potential anticancer drugs

Spectroscopic and rapid kinetic experiments were performed to detail the interaction of human glutathione S-transferases GSTA1-1, GSTM2-2, and GSTP1-1 with 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX). This compound is a representative molecule of a new class of 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives (non-GSH peptidomimetic compounds) that have been designed both to give strong GST inhibition and to accumulate in tumor cells avoiding the extrusion mechanisms mediated by the multidrug resistance protein pumps. We have recently shown that submicromolar amounts of NBDHEX trigger apoptosis in several human tumor cell lines through the dissociation of the JNK.GSTP1-1 complex (Turella, P., Cerella, C., Filomeni, G., Bullo, A., De Maria, F., Ghibelli, L., Ciriolo, M. R., Cianfriglia, M., Mattei, M., Federici, G., Ricci, G., and Caccuri, A. M. (2005) Cancer Res. 65, 3751-3761). Results reported in the present study indicated that NBDHEX behaves like a suicide inhibitor for GSTs. It bound to the H-site and was conjugated with GSH forming a sigma complex at the C-4 of the benzoxadiazole ring. This complex was tightly stabilized in the active site of GSTP1-1 and GSTM2-2, whereas in GSTA1-1 the release of the 6-mercapto-1-hexanol from the sigma complex was the favored event. Docking studies demonstrated the likely localization of the sigma complex in the GST active sites and provide a structural explanation for its strong stabilization.     

Changes of enzyme activity in lipid signaling pathways related to substrate reordering

The static fluid mosaic model of biological membranes has been progressively complemented by a dynamic membrane model that includes phospholipid reordering in domains that are proposed to extend from nanometers to microns. Kinetic models for lipolytic enzymes have only been developed for homogeneous lipid phases. In this work, we develop a generalization of the well-known surface dilution kinetic theory to cases where, in a same lipid phase, both domain and nondomain phases coexist. Our model also allows understanding the changes in enzymatic activity due to a decrease of free substrate concentration when domains are induced by peptides. This lipid reordering and domain dynamics can affect the activity of lipolytic enzymes, and can provide a simple explanation for how basic peptides, with a strong direct interaction with acidic phospholipids (such as beta-amyloid peptide), may cause a complex modulation of the activities of many important enzymes in lipid signaling pathways.     

1H-NMR study of the effect of temperature through reversible unfolding on the heme pocket molecular structure and magnetic properties of aplysia limacina cyano-metmyoglobin

Two-dimensional 1H NMR spectroscopy over a range of temperature through thermal unfolding has been applied to the low-spin, ferric cyanide complex of myoglobin from Aplysia limacina to search for intermediates in the unfolding and to characterize the effect of temperature on the magnetic properties and electronic structure of the heme iron. The observation of strictly linear behavior from 5 to 80 C degrees through the unfolding transition for all hyperfine-shifted resonances indicates the absence of significant populations of intermediate states to the cooperative unfolding with Tm approximately 80 degrees C. The magnetic anisotropies and orientation of the magnetic axes for the complete range of temperatures were also determined for the complex. The anisotropies have very similar magnitudes, and exhibit the expected characteristic temperature dependence, previously observed in the isoelectronic sperm whale myoglobin complex. In contrast to sperm whale Mb, where the orientation of the magnetic axis was completely temperature-independent, the tilt of the major magnetic axis, which correlates with the Fe-CN tilt, decreases at high temperature in Aplysia limacina Mb, indicating a molecular structure that is conserved with temperature, although more plastic than that of sperm whale Mb. The pattern of contact shifts reflects a conserved Fe-His(F8) bond and pi-spin delocalization into the heme, as expected for the orientation of the axial His imidazole.     

Ganglioside-induced differentiation associated protein 1 is a regulator of the mitochondrial network: new implications for Charcot-Marie-Tooth disease

Mutations in GDAP1 lead to severe forms of the peripheral motor and sensory neuropathy, Charcot-Marie-Tooth disease (CMT), which is characterized by heterogeneous phenotypes, including pronounced axonal damage and demyelination. We show that neurons and Schwann cells express ganglioside-induced differentiation associated protein 1 (GDAP1), which suggest that both cell types may contribute to the mixed features of the disease. GDAP1 is located in the mitochondrial outer membrane and regulates the mitochondrial network. Overexpression of GDAP1 induces fragmentation of mitochondria without inducing apoptosis, affecting overall mitochondrial activity, or interfering with mitochondrial fusion. The mitochondrial fusion proteins, mitofusin 1 and 2 and Drp1(K38A), can counterbalance the GDAP1-dependent fission. GDAP1-specific knockdown by RNA interference results in a tubular mitochondrial morphology. GDAP1 truncations that are found in patients who have CMT are not targeted to mitochondria and have lost mitochondrial fragmentation activity. The latter activity also is reduced strongly for disease-associated GDAP1 point mutations. Our data indicate that an exquisitely tight control of mitochondrial dynamics, regulated by GDAP1, is crucial for the proper function of myelinated peripheral nerves.     

Molecular cloning and characterization of an almond 9-hydroperoxide lyase, a new CYP74 targeted to lipid bodies

Oxylipin metabolism represents one of many defence mechanisms employed by plants. It begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases to form fatty acid hydroperoxides that are substrates for several enzymes, including specialized cytochrome P450s known as CYP74s. The targeting of a new CYP74, a 9-hydroperoxide lyase (HPL) from almonds, to the endomembrane system and lipid bodies, both as enzyme activity in almond seeds and as GFP fusions transiently expressed in tobacco protoplasts, is described. Such association of a CYP74 with lipid bodies has not been reported previously. Also described are the properties of a 9-HPL gene, the developmental regulation of its expression, the production and characterization of recombinant 9-HPL in Escherichia coli, and the developmental correlation between gene expression, enzyme activity, and the appearance of volatile C9 aldehydes from HPL action.     

Porin and cytochrome oxidase containing contact sites involved in the oxidation of cytosolic NADH

Cytochrome c (cyto-c) added to isolated mitochondria promotes the oxidation of extra-mitochondrial NADH and the reduction of molecular oxygen associated to the generation of an electrochemical membrane potential available for ATP synthesis. The electron transport pathway activated by exogenous cyto-c molecules is completely distinct from the one catalyzed by the respiratory chain. Dextran sulfate (500 kDa), known to interact with porin (the voltage-dependent anion channel), other than to inhibit the release of ATP synthesized inside the mitochondria, greatly decreases the activity of exogenous NADH/cyto-c system of intact mitochondria but has no effect on the reconstituted system made of mitoplasts and external membrane preparations. The results obtained are consistent with the existence of specific contact sites containing cytochrome oxidase and porin, as components of the inner and the outer membrane respectively, involved in the oxidation of cytosolic NADH. The proposal is put forward that the bi-trans-membrane electron transport chain activated by cytosolic cyto-c becomes, in physio-pathological conditions: (i) functional in removing the excess of cytosolic NADH; (ii) essential for cell survival in the presence of an impairment of the first three respiratory complexes; and (iii) an additional source of energy at the beginning of apoptosis.