Deletion of naive CD8 T cells requires persistent antigen and is not programmed by an initial signal from the tolerogenic APC

Activation of naive CD8 T cells in vivo requires the recognition of cognate peptide-MHC complexes on APCs. Depending upon the activation status of the APC, such recognition will promote either a vigorous immune response or T cell tolerance and deletion. Recent studies suggest that the initial signals provided by APCs are sufficient to program the proliferation of naive CD8 T cells and their differentiation into effector cells. In this study, we sought to determine whether an initial encounter with tolerogenic APCs was sufficient to program deletion of naive CD8 T cells. Surprisingly, we find that regardless of whether naive CD8 T cells were stimulated by activated or quiescent APCs, transfer of the activated T cells into an Ag-free host was sufficient to ensure survival. Thus, although the extent of clonal expansion and development of effector function is determined by the activation status of the stimulatory APC, peripheral clonal deletion requires persistent Ag and is not determined by the initial stimulatory event.     

Influence of the solvent ability to form hydrogen bonds in the crystal structure of ([3β,5β,7α,12α]-3[(norbornyl-2-acetyl)-amino]-7,12-dihydroxycholan-24-oic acid (a norbornyl derivative of cholic acid)

A norbornyl-2-acetyl derivative of cholic acid ([3β,5β,7α,12α]-3[(norbornyl-2-acetyl)-amino]-7,12-dihydroxycholan-24-oic acid -NbCH₂CA-) was synthesized and recrystallized in two dipolar aprotic solvents (acetone, DMSO) and in one protic solvent (2-propanol). In DMSO and acetone the crystals are orthorhombic, P2₁2₁2₁ (all their parameters being very similar) while in 2-propanol the crystal is monoclinic, P2₁. The inclusion complexes with the solvent have a 1:1 stochiometry with DMSO and acetone and 1:2 with 2-propanol. All solvents are forming a hydrogen bond with the amide bond of the bridge between the norbornyl residue and the steroid nucleus of the bile acid. In DMSO and acetone the β side of the steroid groups lies in the same region facilitating hydrophobic interactions, and the molecules are disposed in an antiparallel orientation (the methyl groups having a β interdigitation) forming bilayers. The width of the bilayers is 9.231 Å and 8.859 Å in DMSO and acetone, respectively. A lamellar structure is also evident for the crystal in 2-propanol (the width being 11.908 Å), but the packing is different from the previous one since a sliding between the steroid groups is observed and the methyl groups are not interdigitated. Four different hydrogen bonds are established by every steroid molecule in the NbCH₂CA/DMSO (or acetone) crystal. This hydrogen bond network interconnects the hydrophilic regions of the lamellar structure. The hydrogen bond network of the NbCH₂CA:2-propanol crystal is different because of the different abilities of 2-propanol to form hydrogen bonds. The side chain has a ttti conformation in the two orthorhombic crystals, and a tgtg one in the monoclinic crystal.     

Solanum Tuber-bearing Species Resistance Behavior Against Nacobbus aberrans

Naccobus aberrans is a major pest of the potato crop in the Andean regions of Argentina, Bolivia, and Perú. It is endemic in northwest Argentina and is also found in lowlands. The resistance of eleven Andean potato landraces and three accessions of the wild tuber-bearing species Solanum acaule, S. infundibuliforme, and S. megistacrolobum were evaluated against a population of N. aberrans from Coctaca, Jujuy province, while Solanum tuberosum ssp. tuberosum 'Spunta', 'Kennebec', and 'Frital INTA' were evaluated against a population from the southeast of Buenos Aires province. The presence, the number of galls, and the number of individuals were recorded. In addition, a reproduction factor was calculated and races were determined. Results showed that the N. aberrans population from Coctaca corresponded to race 2 and the population from the lowlands belonged to the sugar beet group. Landrace Azul, one genotype of S. megistacrolobum, and two genotypes of S. acaule showed resistance towards the race from Coctaca while no infection was recorded in potato cultivars with the Naccobus race from the lowland area.     

Topoisomerase II, not topoisomerase I, is the proficient relaxase of nucleosomal DNA

Eukaryotic topoisomerases I and II efficiently remove helical tension in naked DNA molecules. However, this activity has not been examined in nucleosomal DNA, their natural substrate. Here, we obtained yeast minichromosomes holding DNA under (+) helical tension, and incubated them with topoisomerases. We show that DNA supercoiling density can rise above +0.04 without displacement of the histones and that the typical nucleosome topology is restored upon DNA relaxation. However, in contrast to what is observed in naked DNA, topoisomerase II relaxes nucleosomal DNA much faster than topoisomerase I. The same effect occurs in cell extracts containing physiological dosages of topoisomeraseI and II. Apparently, the DNA strand-rotation mechanism of topoisomerase I does not efficiently relax chromatin, which imposes barriers for DNA twist diffusion. Conversely, the DNA cross-inversion mechanism of topoisomerase II is facilitated in chromatin, which favor the juxtaposition of DNA segments. We conclude that topoisomerase II is the main modulator of DNA topology in chromatin fibers. The nonessential topoisomerase I then assists DNA relaxation where chromatin structure impairs DNA juxtaposition but allows twist diffusion.     

Trypanosoma cruzi contains a single detectable uracil-DNA glycosylase and repairs uracil exclusively via short patch base excision repair

Enzymes involved in genomic maintenance of human parasites are attractive targets for parasite-specific drugs. The parasitic protozoan Trypanosoma cruzi contains at least two enzymes involved in the protection against potentially mutagenic uracil, a deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and a uracil-DNA glycosylase belonging to the highly conserved UNG-family. Uracil-DNA glycosylase activities excise uracil from DNA and initiate a multistep base-excision repair (BER) pathway to restore the correct nucleotide sequence. Here we report the biochemical characterisation of T.cruzi UNG (TcUNG) and its contribution to the total uracil repair activity in T.cruzi. TcUNG is shown to be the major uracil-DNA glycosylase in T.cruzi. The purified recombinant TcUNG exhibits substrate preference for removal of uracil in the order ssU>U:G>U:A, and has no associated thymine-DNA glycosylase activity. T.cruzi apparently repairs U:G DNA substrate exclusively via short-patch BER, but the DNA polymerase involved surprisingly displays a vertebrate POLdelta-like pattern of inhibition. Back-up UDG activities such as SMUG, TDG and MBD4 were not found, underlying the importance of the TcUNG enzyme in protection against uracil in DNA and as a potential target for drug therapy.     

Distribution of CK2, its substrate MAP1B and phosphatases in neuronal cells

Neuronal morphogenesis depends on the organization of cytoskeletal elements among which microtubules play a very important role. The organization of microtubules is controlled by the presence of microtubule-associated proteins (MAPs), the activity of which is modulated by phosphorylation and dephosphorylation. One of these MAPs is MAP1B, which is very abundant within growing axons of developing neurons where it is found phosphorylated by several protein kinases including CK2. The expression of MAP1B is notably decreased after neuronal maturation in parallel with a change in the localization of the protein, which becomes largely concentrated in neuronal cell bodies and dendrites. Interestingly, MAP1B remains highly phosphorylated at sites targeted by protein kinase CK2 in mature neurons.We have analyzed the expression and localization of CK2 catalytic subunits along neuronal development. CK2 subunit appears early during development whereas CK2 subunit appears within mature neurons at the time of dendrite maturation and synaptogenesis, in parallel with the change in the localization of MAP1B. CK2 subunit is found associated with microtubule preparations obtained from either grey matter or white matter from adult bovine brain, whereas CK2 subunit is highly enriched in microtubules obtained from grey matter. These results lend support to the hypothesis that CK2 subunit is concentrated in neuronal cell bodies and dendrites, where it associates with microtubules, thus contributing to the increased phosphorylation of MAP1B in this localization in mature neurons.     

The effects of drying on sediment nitrogen content in a Mediterranean intermittent stream: a microcosms study

Mediterranean climates predispose aquatic systems to both flood and drought periods, therefore, stream sediments may be exposed to desiccation periods. Changes in oxygen concentrations and sediment water content influence the biotic processes implicated in nitrogen dynamics. The objectives of this study were to identify (1) the changes of inorganic nitrogen in stream sediments during the transition from wet to dry conditions, and (2) the underlying processes in N dynamics and its regulation. Extractable sediment NO₃ ⁻-N and NH₄ ⁺-N, organic matter and extractable organic carbon content were assessed during natural desiccation in microcosms with sediments from an intermittent Mediterranean stream. In agreement with our initial hypothesis, our results showed how the NO₃ ⁻-N content of the sediment was enhanced during the first 10 days of sediment drying, whereas NH₄ ⁺-N was lost by 14 days post-drying. During the first 10 days, sediment desiccation seemed to stimulate the net N-mineralization and net nitrification from sediments. Afterwards, the extractable NO₃ ⁻-N concentration sharply dropped, which may be attributed to lower ammonium-oxidation rates as ammonium and organic matter are depleted, and to an increase in NO₃ ⁻-N consumption by microbial populations. Denitrification was inhibited, with a significant decrease as % water-filled pore space lowered. We hypothesize that the sediment inorganic N content enhanced during sediment desiccation could be released as part of the N pulse observed after sediment rewetting. However, the stream N availability after rewetting dried sediments would differ depending on desiccation period duration.     

Adenylate Uptake by Proplastids from Cultured Cells of Tobacco {Nicotiana tabacum L. cv. BY2) Indicates That an Adenylate Translocator is Present in All Types of Plastid

The presence of an adenylate translocator in the envelope membranesof proplastids isolated from the cultured cells of tobacco (Nicotianatabacum L. cv. BY2) was examined by means of transport experimentsusing the silicone oil filtering centrifugation technique. Itwas observed that proplastids can import [³H]ATP, [³H]ADP, [³H]AMPand less specifically ADP-[¹⁴C]Glc which can eventually be usedfor starch biosynthesis. The effects of specific inhibitorsof the mitochondrial adenylate translocator, i.e. atractyloside,bongkrekic acid and carboxyatractyloside were tested. Similarto the case of amyloplasts isolated from the cultured cellsof sycamore and chloroplasts isolated from spinach leaves, onlyATP and ADP-Glc uptake were shown to be partially inhibitedby carboxyatractyloside. On the other hand, neither atractylosidenor bongkrekic acid exerted a significant inhibitory effecton adenylate uptake. (Received August 8, 1992; Accepted November 26, 1992)     

Modular glucuronoxylan-specific xylanase with a family CBM35 carbohydrate-binding module

Xyn30D from the xylanolytic strain Paenibacillus barcinonensis has been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing a K(m) of 14.72 mg/ml and a k(cat) value of 1,510 min(-1). The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)(8) barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.     

Impact of Daily Thermocycles on Hatching Rhythms,Larval Performance and Sex Differentiation of Zebrafish

In the wild, water temperature cycles daily: it warms up after sunrise, and cools rapidly after sunset. Surprisingly, the impact of such daily thermocycles during the early development of fish remains neglected. We investigated the influence of constant vs daily thermocycles in zebrafish, from embryo development to sexual differentiation, by applying four temperature regimens: two constant (24°C and 28°C) and two daily thermocycles: 28:24°C, TC (thermophase coinciding with daytime, and cryophase coinciding with night-time) and 24:28°C, CT (opposite to TC) in a 12:12 h light:dark cycle (LD). Embryo development was temperature-dependent but enhanced at 28°C and TC. Hatching rhythms were diurnal (around 4 h after lights on), but temperature- and cycle-sensitive, since hatching occurred sooner at 28°C (48 hours post fertilization; hpf) while it was delayed at 24°C (96 hpf). Under TC, hatching occurred at 72 hpf, while under CT hatching displayed two peaks (at 70 hpf and 94 hpf). In constant light (LL) or darkness (DD), hatching rhythms persisted with tau close to 24 h, suggesting a clock-controlled “gating” mechanism. Under 28°C or TC, larvae showed the best performance (high growth and survival, and low malformations). The sex ratio was strongly influenced by temperature, as the proportion of females was higher in CT and TC (79 and 83% respectively), contrasting with 28°C and 24°C, which led to more males (83 and 76%). Ovarian aromatase (cyp19a) expression in females was highest in TC and CT (6.5 and 4.6 fold higher than at 28°C, respectively); while anti-müllerian hormone (amh) expression in males increased in testis at 24°C (3.6 fold higher compared to TC) and particularly at 28°C (14.3 fold increase). Taken together, these findings highlight the key role of environmental cycles during early development, which shaped the daily rhythms in fish embryo and larvae, and ultimately influenced sex differentiation.