IL-7 overexpression in transgenic mouse keratinocytes causes a lymphoproliferative skin disease dominated by intermediate TCR cells: evidence for a hierarchy in IL-7 responsiveness among cutaneous T cells

IL-7 is a keratinocyte-derived lymphocyte growth factor critical for the development of gammadelta T cells including murine dendritic epidermal T cells (DETC). We derived transgenic mice that overexpress IL-7 in basal keratinocytes under the control of the human K14 promoter. These K14/IL-7 mice develop dermal and epidermal T cell infiltrates associated with alopecia. This lymphoproliferative skin disease is substantially more severe in mice homozygous for the K14/IL-7 transgene. Conventional DETC expressing a Vgamma5 Vdelta1 TCR are rare or absent among the cutaneous T cells in these mice. The T cells in the skin infiltrates of young K14/IL-7 mice are predominantly gammadelta T cells that express intermediate levels of TCR, are negative for E-cadherin, often lack expression of CD2, and include cells that coexpress NK1.1. T cells expressing intermediate levels of a TCR-alphabeta are also present in transgenic skin, and progressively increase in number as the mice age. Phenotypically similar intermediate gammadelta and alphabeta T cell subsets also constitute the major lymphocyte populations recovered from organ culture of normal mouse skin in the presence of IL-7, suggesting that the T cells that accumulate in the epidermis of K14/IL-7 mice are derived from precursors normally resident in skin. We conclude that intermediate TCR cells, some of which coexpress NK1.1, can be selectively expanded in skin under the influence of IL-7 produced locally. Our results also suggest that features of the epidermal microenvironment besides keratinocyte-derived IL-7 account for the normal predominance of Vgamma5 Vdelta1 DETC in mouse epidermis.     

New Octopus-like Phthalocyanines as Fullerene Receptors: Synthesis and Photophysical Investigation

Octopus-like zinc and magnesium phthalocyaninates bearing eight flexible benzylated diethylene glycol chains were synthesized and their interaction with fullerenes C₆₀ and C₇₀ was investigated by UV-Vis spectrophotometric titration, as well as by steady-state and time-resolved fluorescence spectroscopy in chloroform and toluene media. These measurements revealed a high affinity of receptors for C₆₀ and C₇₀, with selectivity to C₇₀: binding constants for C₇₀ are almost two times higher than for C₆₀. These results are interpreted by means of quantum-chemical calculations using the PM6-DH2 Hamiltonian. The binding constants also depend on both the nature of the metal ion in the receptor and the solvent. It is expected that the obtained molecules and supramolecular complexes can be used for further elaboration of optoelectronic donor-acceptor materials.     

Electrochemical properties of Pb–Sb, Pb–Ca–Sn and Pb–Co3O4 anodes in copper electrowinning

Three types of anode, Pb–Sb, Pb–Ca–Sn and Pb–Co₃O₄, for copper electrowinning were investigated. The corrosion resistance, as evaluated by cyclic voltammetric (CV) measurements was higher for Pb–Co₃O₄ than for Pb–Sb and Pb–Ca–Sn. During prolonged electrowinning under galvanostatic conditions, the anodic reaction on the Pb–Co₃O₄ anode was depolarized by 0.053 V as compared to Pb–Sb, and by 0.106 V with respect to Pb–Ca–Sn. The composition and structure of the anodic layer were determined by XPS, X-ray and SEM analyses. The surface layer on the three anodes examined was composed mainly of PbSO₄, -PbO₂ and -PbO₂. Different structure of the surface layer was observed: loose and highly spread coral-like structure in the case of Pb–Sb; fibrous structure in the case of Pb–Ca–Sn and dense, fine-grained structure in the case of Pb–Co₃O₄.     

Impact of A134 and E218 Amino Acid Residues of Tropomyosin on Its Flexibility and Function

Tropomyosin (Tpm) is one of the major actin-binding proteins that play a crucial role in the regulation of muscle contraction. The flexibility of the Tpm molecule is believed to be vital for its functioning, although its role and significance are under discussion. We choose two sites of the Tpm molecule that presumably have high flexibility and stabilized them with the A134L or E218L substitutions. Applying differential scanning calorimetry (DSC), molecular dynamics (MD), co-sedimentation, trypsin digestion, and in vitro motility assay, we characterized the properties of Tpm molecules with these substitutions. The A134L mutation prevented proteolysis of Tpm molecule by trypsin, and both substitutions increased the thermal stability of Tpm and its bending stiffness estimated from MD simulation. None of these mutations affected the primary binding of Tpm to F-actin; still, both of them increased the thermal stability of the actin-Tpm complex and maximal sliding velocity of regulated thin filaments in vitro at a saturating Ca²⁺ concentration. However, the mutations differently affected the Ca²⁺ sensitivity of the sliding velocity and pulling force produced by myosin heads. The data suggest that both regions of instability are essential for correct regulation and fine-tuning of Ca²⁺-dependent interaction of myosin heads with F-actin.     

Mutant PCNA alleles are associated with cdc phenotypes and sensitivity to DNA damage in fission yeast

Twenty-eight site-directed mutations were introduced into the fission yeast gene (pcn1+) that encodes proliferating cell nuclear antigen (PCNA) and their in vivo effects analyzed in a strain with a null pcn1 background. Mutants defective in enhancing processivity of DNA polymerase delta have previously been identified. In this study, we assessed all of the mutants for their sensitivities to temperature, hydroxyurea, UV irradiation and methyl methanesulfonate (MMS), and specific mutants were also tested for sensitivity to gamma irradiation. One cold-sensitive allele, pcn1-3, was characterized in detail. This mutant had a recessive cold-sensitive cdc phenotype and showed sensitivity to hydroxyurea, UV, and gamma irradiation. At the non-permissive temperature pcn1-3 protein was able to form homotrimers in solution and showed increased stimulation of both synthetic activity and processivity of DNA polymerase delta relative to the wild-type Pcn1+ protein. Epistasis analyses indicated that pcn1-3 is defective in the repair pathway involving rad2+ but not defective in the classical nucleotide excision repair pathway involving rad13+. Furthermore, pcn1-3 is either synthetically or conditionally lethal in null checkpoint rad backgrounds and displays a mitotic catastrophe phenotype in these backgrounds. A model for how pcn1-3 defects may affect DNA repair and replication is presented.     

Magnetic resonance images of neuronal migration anomalies

Neuronal migration anomalies are a spectrum of brain malformations caused by insults to migrating neuroblasts during the sixth week to fifth month of gestation. To study the characteristics of MRI findings in migration anomalies, MR images of 36 patients (28 children and 8 adults) with migration anomalies were evaluated. Five patients had lissencephaly, eight had pachygyria, twelve had schizencephaly, six had heterotopias of gray matter, three had hemimegalencephaly, and two had polymicrogyria. The frequency of migration anomalies was 0.51% of all cranial MRI studies and 1.21% of pediatric cranial MRI studies at our hospital. The major clinical presentations of these patients were seizure (64%), development delay (42%), motor deficits (42%) and mental retardation (25%). Twenty-five patients (69%) associated with other brain anomalies, including: other migration anomalies in 12 cases (33%), absence of the septum pellucidum in 10 cases (28%), Dandy-Walker malformation/variant in 5 cases, arachnoid cyst in 4 cases, agenesis of the corpus callosum in 3 cases, holoprosencephaly in 2 cases, mega cisterna magna in 1 case and cephalocele in 1 case. Some of them presented with multiple complicated anomalies. As MR imaging provides superb gray-white matter distinction, details of cortical anatomy and multiplanar capability, it can clearly delineate the detail morphologic changes of the brain caused by neuronal migration disorders as well as the associated anomalies.     

Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites

Some cytochrome P450 catalyzed reactions show atypical kinetics, and these kinetic processes can be grouped into five categories: activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. A two-site model in which the enzyme can bind two substrate molecules simultaneously is presented which can be used to describe all of these observed kinetic properties. Sigmoidal kinetic characteristics were observed for carbamazepine metabolism by CYP3A4 and naphthalene metabolism by CYPs 2B6, 2C8, 2C9, and 3A5 as well as dapsone metabolism by CYP2C9. Naphthalene metabolism by CYP3A4 and naproxen metabolism by CYP2C9 demonstrated nonhyperbolic enzyme kinetics suggestive of a low Km, low Vmax component for the first substrate molecule and a high Km, high Vmax component for the second substrate molecule. 7, 8-Benzoflavone activation of phenanthrene metabolism by CYP3A4 and dapsone activation of flurbiprofen and naproxen metabolism by CYP2C9 were also observed. Furthermore, partial inhibition of 7, 8-benzoflavone metabolism by phenanthrene was observed. These results demonstrate that various P450 isoforms may exhibit atypical enzyme kinetics depending on the substrate(s) employed and that these results may be explained by a model which includes simultaneous binding of two substrate molecules in the active site.