CD5-Dependent CK2 Activation Pathway Regulates Threshold for T Cell Anergy

CD5 activates casein kinase 2 (CK2), a serine/threonine kinase that constitutively associates with the CK2-binding domain at the end of its cytoplasmic tail. To determine the physiological significance of CD5-dependent CK2 activation in T cells, we generated a knock-in mouse that expresses a CD5 protein containing a microdeletion with selective inability to interact with CK2 (CD5ΔCK2BD). The levels of CD5 on developing and mature T cell populations from CD5ΔCK2BD mice and CD5 wild-type (WT) mice were similar. The thymus of CD5ΔCK2BD mice contained fewer double-positive thymocytes than did that of both CD5WT and CD5 knockout (KO) mice, although the numbers of all other immature and mature T cell populations were unaltered. CD5ΔCK2BD T cells hypoproliferated and exhibited enhanced activation-induced cell death when stimulated with anti-CD3 or cognate peptide in comparison with CD5WT T cells. We also found that functional CD5-dependent CK2 signaling was necessary for efficient differentiation of naive CD4(+) T cells into Th2 and Th17 cells, but not Th1 cells. We previously showed that experimental autoimmune encephalomyelitis (EAE) in CD5KO mice was less severe and delayed in onset than in CD5WT mice. Remarkably, CD5ΔCK2BD mice recapitulated both EAE severity and disease onset of CD5KO mice. Increasing the immunization dose of myelin oligodendrocyte glycoprotein 35-55 peptide, a model that mimics high-dose tolerance, led to decreased severity of EAE in CD5WT mice but not in CD5KO or CD5ΔCK2BD mice. This property was recapitulated in in vitro restimulation assays. These results demonstrate that CD5-CK2 signaling sets the threshold for T cell responsiveness and is necessary for efficient generation of Th2 and Th17 cells.     

Micro- and macromethod assays for the ecological study of Legionella pneumophila

The survival of a strain of Legionella pneumophila (Lp-1) inoculated in artificial water microcosms was investigated with and without an amoebal host and varying environmental conditions, such as biofilm formation, amount of nutrients and incubation temperature. The results obtained using short (micromethod) and long (macromethod) term methods showed that L. pneumophila Lp-1 dies rapidly at 4 degrees C in the "macromethod" assay. When the same temperature (4 degrees C) was applied to the "micromethod" assay, L. pneumophila Lp-1 survived for three weeks, although it progressively decreased. At an incubation temperature of 30 degrees C, the aquatic environment was more favourable and better survival emerged in the "macromethod"; in contrast, this favourable temperature condition did not improve the survival of L. pneumophila Lp-1 cultured with the "micromethod". The role of the protozoa Acanthamoeba polyphaga proved to be indispensable for legionella survival only when environmental conditions become unfavourable.     

Mutations in the Small GTPase Gene RAB39B Are Responsible for X-linked Mental Retardation Associated with Autism, Epilepsy, and Macrocephaly

Human Mental Retardation (MR) is a common and highly heterogeneous pediatric disorder affecting around 3% of the general population; at least 215 X-linked MR (XLMR) conditions have been described, and mutations have been identified in 83 different genes, encoding proteins with a variety of function, such as chromatin remodeling, synaptic function, and intracellular trafficking. The small GTPases of the RAB family, which play an essential role in intracellular vesicular trafficking, have been shown to be involved in MR. We report here the identification of mutations in the small GTPase RAB39B gene in two male patients. One mutation in family X (D-23) introduced a stop codon seven amino acids after the start codon (c.21C > A; p.Y7X). A second mutation, in the MRX72 family, altered the 5′ splice site (c.215+1G > A) and normal splicing. Neither instance produced a protein. Mutations segregate with the disease in the families, and in some family members intellectual disabilities were associated with autism spectrum disorder, epileptic seizures, and macrocephaly. We show that RAB39B, a novel RAB GTPase of unknown function, is a neuronal-specific protein that is localized to the Golgi compartment. Its downregulation leads to an alteration in the number and morphology of neurite growth cones and a significant reduction in presynaptic buttons, suggesting that RAB39B is required for synapse formation and maintenance. Our results demonstrate developmental and functional neuronal alteration as a consequence of downregulation of RAB39B and emphasize the critical role of vesicular trafficking in the development of neurons and human intellectual abilities.     

Combined effects of radiotherapy and photodynamic therapy on an in vitro human prostate model

Human prostate cancer cells were evaluated for growth after photodynamic therapy, radiotherapy, and combined treatment. Indocyanine green was tested as a photosensitizer and radiosensitizer. Two human cell lines were used: PC-3 derived from prostate carcinoma, and EPN derived from normal prostate tissue. The light source used for the photoactivation experiments was a diode laser peaked at 805 nm. The light dose incident on cells was 108 J/cm(2). Ionizing radiation was produced by a linear accelerator, and the dose was 2, 4 and 6 Gy. Cytotoxicity was evaluated by measuring the colony forming ability of cells. Our results show that indocyanine green induces cell death by photoactivation, but it does not act as a radiosensitizer if used with ionizing radiation. The combined treatment of photodynamic therapy and radiotherapy produces an additive effect which does not depend on the sequence of the two treatments. Combined treatments could be more useful since they allow the reduction of the ionizing radiation dose to obtain the same effect as one obtainable by radiotherapy alone.     

Iron Release in Erythrocytes and Plasma Non Protein-bound Iron in Hypoxic and Non Hypoxic Newborns

Iron is released in a desferrioxamine (DFO)-chelatable form (DCI) when erythrocytes are challenged by an oxidative stress. In &#103 -thalassemic erythrocytes, both DCI content and release (after aerobic incubation for 24 h) are increased and correlated with the fetal hemoglobin (HbF) levels. Since erythrocytes from newborns have an extremely high content of HbF and are exposed to conditions of oxidative stress, the release of iron in these erythrocytes was investigated. The erythrocyte DCI content was increased in preterm but not in term newborns as compared to adults, while the release was increased in both preterm and term erythrocytes. The level of plasma non protein-bound iron (NPBI), which was not detectable in adults, was much higher in preterm than in term newborns. When term plus preterm newborns were divided in two groups, normoxic and hypoxic, according to cord blood pH, it was found that both iron release and NBPI were markedly higher in the hypoxic newborns compared to normoxic ones. Similar results were also obtained when the preterm and term infants were considered separately on the basis of cord blood pH. Therefore, iron release and NPBI are higher when conditions of hypoxia occur. In fact, when the values for iron release and NPBI were separately plotted against cord blood pH values, significant negative correlations were seen in both cases. NPBI was correlated with iron release seen in all the newborns and a significant part of the released iron could be recovered into the incubation medium at the end of the incubation.     

De novo root formation in thin cell layers of tobacco: changes in free and bound polyamines

Thin cell layers excised from tobacco ( Nicotiana tabacum L. cv. Samsun) stem internodes, with an appropriate exogenous hormonal balance, were able to form a greater number of roots, and in a larger percentage of the explants (93%) than when they were excised from pedicels (40%). The developmental sequence of root formation and explant growth were followed by histological analysis. Free and bound [trichloroacetic acid (TCA)-soluble and -insoluble] putrescine and spermidine increased in the explants, particularly when root meristemoids appeared. These meristemoids originated in the superficial (day 6 in culture) or deep (days 10–11) layers and inside the newly formed callus (day 25). At those times, TCA-soluble and, to a lesser extent, TCA-insoluble bound putrescine predominated over the other polyamines. Spermine was always present in trace amounts. Polyamines decreased again when root and callus formation was completed (day 30). The involvement of these three classes of polyamines (free, TCA-soluble and -insoluble) in morphogenic processes is discussed.     

Metabolic reduction of chromium,as related to its carcinogenic properties

At variance with Cr(III), Cr(VI) compounds easily cross cell membranes and exert genotoxic effects. No metabolic oxidation of Cr(III) could be detected, whereas Cr(VI) reduction was observed in the presence of body fluids and subcellular fractions of various tissues from several animal species. The differential efficiency of this process may account for the selection of target tissues in Cr(VI) carcinogenesis. For instance, reduction by saliva and gastric juice may explain a lack of carcinogenicity by the oral route; reduction inside erythrocytes may explain a lack of carcinogenicity at a distance from administration sites; reduction by the epithelial-lining fluid of terminal airways and by alveolar macrophages may be consistent with the occurrence of thresholds in lung carcinogenesis. Liver preparations displayed the top efficiency in reducing Cr(VI), whereas skeletal muscle, i.e., a typical target in experimental Cr(VI) carcinogenesis, had no detectable activity. Bronchial tree and peripheral lung parenchyma preparations from almost 100 individuals reduced Cr(VI) to a variable extent. The efficiency of lung parenchyma and of isolated alveolar macrophages was enhanced in cigarette smokers. In rats, Cr(VI) reduction by lung preparations was significantly stimulated by the repeated i.t. instillation of Cr(VI) itself. Among the electron donors (chiefly GSH) and enzymatic mechanisms responsible for the intracellular Cr(VI) reduction, such as cytochrome P-450 reductase, glutathione redactase, and aldehyde oxidase, an important role can be ascribed to cytosolic DT diaphorase activity, usually catalyzing a 2-electron reduction.     

Specific protein changes contribute to the differential muscle mass loss during ageing

In the skeletal muscle, the ageing process is characterized by a loss of muscle mass and strength, coupled with a decline of mitochondrial function and a decrease of satellite cells. This profile is more pronounced in hindlimb than in forelimb muscles, both in humans and in rodents. Utilizing light and electron microscopy, myosin heavy chain isoform distribution, proteomic analysis by 2D‐DIGE, MALDI‐TOF MS and quantitative immunoblotting, this study analyzes the protein levels and the nuclear localization of specific molecules, which can contribute to a preferential muscle loss. Our results identify the molecular changes in the hindlimb (gastrocnemius) and forelimb (triceps) muscles during ageing in rats (3‐ and 22‐month‐old). Specifically, the oxidative metabolism contributes to tissue homeostasis in triceps, whereas respiratory chain disruption and oxidative‐stress‐induced damage imbalance the homeostasis in gastrocnemius muscle. High levels of dihydrolipoyllysine‐residue acetyltransferase (Dlat) and ATP synthase subunit alpha (Atp5a1) are detected in triceps and gastrocnemius, respectively. Interestingly, in triceps, both molecules are increased in the nucleus in aged rats and are associated to an increased protein acetylation and myoglobin availability. Furthermore, autophagy is retained in triceps whereas an enhanced fusion, decrement of mitophagy and of regenerative potential is observed in aged gastrocnemius muscle.     

Influence of aperiodic summer droughts on leaf litter breakdown and macroinvertebrate assemblages: testing the <Emphasis Type="Italic">drying memory</Emphasis> in a Central Apennines River (Aterno River,Italy)

Management of multiple exploited stocks of anadromous salmonids in large catchments requires understanding of movement and catchment use by the migrating fish and of their harvesting. The spawning migration of sea trout (Salmo trutta) and Atlantic salmon (Salmo salar) was studied in the River Tweed, UK, using acoustic telemetry to complement exploitation rate data and to quantify catchment penetration. Salmon (n = 79) and sea trout (n = 65) were tagged in the tidal-influenced Tweed in summer–autumn. No tagged salmon left the river before spawning, but 3% (2010) and 8% (2011) of pre-spawning sea trout dropped out. Combined tag regurgitation/fish mortality in salmon was 12.5%, while trout mortality was 6% (2010) and 0% (2011). The estimated spawning positions of salmon and sea trout differed; tagged salmon were mostly in the main channel while trout occurred mostly in the upper Tweed and tributaries. Early fish migrated upstream slower than later fish, but sea trout moved through the lower-middle river more quickly than salmon, partly supporting the hypothesis that the lower exploitation rate in autumn of trout (1 vs 3.3% for salmon) there is generated by differences in migration behaviour.     

Characterization of a Small Family (CAIII) of Microsatellite-Containing Sequences with X-Y Homology

Four X-linked loci showing homology with a previously described Y-linked polymorphic locus (DYS413) were identified and characterized. By fluorescent in situ hybridization (FISH), somatic cell hybrids, and YAC screening, the X-linked members of this small family of sequences (CAIII) all map in Xp22, while the Y members map in Yq11. These loci contribute to the overall similarity of the two genomic regions. All of the CAIII loci contain an internal microsatellite of the (CA)_n type. The microsatellites display extensive length polymorphism in two of the X-linked members as well as in the Y members. In addition, common sequence variants are found in the portions flanking the microsatellites in two of the X-linked members. Our results indicate that, during the evolution of this family, length variation on the Y chromosome was accumulated at a rate not slower than that on the X chromosome. Finally, these sequences represent a model system with which to analyze human populations for similar X- and Y-linked polymorphisms. Received: 29 July 1996 / Accepted: 15 January 1997