Observations on the behavior of vitreous ice at approximately 82 and approximately 12 K

In an attempt to determine why cooling with liquid helium actually proved disadvantageous in our electron cryotomography experiments, further tests were performed to explore the differences in vitreous ice at approximately 82 and approximately 12 K. Electron diffraction patterns showed clearly that the vitreous ice of interest in biological electron cryomicroscopy (i.e., plunge-frozen, buffered protein solutions) does indeed collapse into a higher density phase when irradiated with as few as 2-3 e-/A2 at approximately 12 K. The high density phase spontaneously expanded back to a state resembling the original, low density phase over a period of hours at approximately 82 K. Movements of gold fiducials and changes in the lengths of tunnels drilled through the ice confirmed these phase changes, and also revealed gross changes in the concavity of the ice layer spanning circular holes in the carbon support. Brief warmup-cooldown cycles from approximately 12 to approximately 82 K and back, as would be required by the flip-flop cryorotation stage, did not induce a global phase change, but did allow certain local strains to relax. Several observations including the rates of tunnel collapse and the production of beam footprints suggested that the high density phase flows more readily in response to irradiation. Finally, the patterns of bubbling were different at the two temperatures. It is concluded that the collapse of vitreous ice at approximately 12 K around macromolecules is too rapid to account alone for the problematic loss of contrast seen, which must instead be due to secondary effects such as changes in the mobility of radiolytic fragments and water.     

CD38 expression enhances sensitivity of lymphoma T and B cell lines to biochemical and receptor-mediated apoptosis

CD38 has been widely characterised both as an ectoenzyme and as a receptor. In the present paper, we investigated the role of CD38 as possible modulator of apoptosis. CD38-positive (CD38(+)) and negative (CD38(-)) fractions, obtained by sorting CD38(+) cells from lymphoma T (Jurkat) and lymphoma B (Raji) and by transfecting lymphoma LG14 and myeloid leukemia K562 cell lines, were used. Cellular subpopulations were exposed to different triggers (H(2)O(2), UV-B, alpha-TOS and hrTRAIL) and the extent of apoptosis was determined by Annexin V-FITC/PI assay. Our data showed that, in lymphoma cells, propensity to apoptosis was significantly linked to CD38 expression and that, remarkably, such response was independent of the nature of the trigger used. Inhibition of CD38 expression by antisense oligonucleotides treatment resulted in CD38-silenced fractions which were as prone to apoptosis as CD38(-) ones. Notably, susceptibility of K562 to apoptosis-inducing challenges was not affected by CD38 expression.     

Memory T(H)2 cells induce alternatively activated macrophages to mediate protection against nematode parasites

Although primary and memory responses against bacteria and viruses have been studied extensively, T helper type 2 (T(H)2) effector mechanisms leading to host protection against helminthic parasites remain elusive. Examination of the intestinal epithelial submucosa of mice after primary and secondary infections by a natural gastrointestinal parasite revealed a distinct immune-cell infiltrate after challenge, featuring interleukin-4-expressing memory CD4(+) T cells that induced IL-4 receptor(hi) (IL-4R(hi)) CD206(+) alternatively activated macrophages. In turn, these alternatively activated macrophages (AAMacs) functioned as important effector cells of the protective memory response contributing to parasite elimination, demonstrating a previously unknown mechanism for host protection against intestinal helminths.     

The apoptosome activates caspase-9 by dimerization

The apical protease of the human intrinsic apoptotic pathway, caspase-9, is activated in a polymeric activation platform known as the apoptosome. The mechanism has been debated, and two contrasting hypotheses have been suggested. One of these postulates an allosteric activation of monomeric caspase-9; the other postulates a dimer-driven assembly at the surface of the apoptosome--the "induced proximity" model. We show that both Hofmeister salts and a reconstituted mini-apoptosome activate caspase-9 by a second-order process, compatible with a conserved dimer-driven process. Significantly, replacement of the recruitment domain of the apical caspase of the extrinsic apoptotic pathway, caspase-8, by that of caspase-9 allows activation of this hybrid caspase by the apoptosome. Consequently, apical caspases can be activated simply by directing their zymogens to the apoptosome, ruling out the requirement for allosteric activation and supporting an induced proximity dimerization model for apical caspase activation in vivo.     

Mosquito transgenesis: what is the fitness cost?

The generation of transgenic mosquitoes with a minimal fitness load is a prerequisite for the success of strategies for controlling mosquito-borne diseases using transgenic insects. It is important to assemble as much information as possible on this subject because realistic estimates of transgene fitness costs are essential for modeling and planning release strategies. Transgenic mosquitoes must have minimal fitness costs, because such costs would reduce the effectiveness of the genetic drive mechanisms that are used to introduce the transgenes into field mosquito populations. Several factors affect fitness of transgenic mosquitoes, including the potential negative effect of transgene products and insertional mutagenesis. Studies to assess fitness of transgenic mosquitoes in the field (as opposed to the laboratory) are still needed.     

Comparative Study of Alkaline, Saline, and Mixed Saline–Alkaline Stresses with Regard to Their Effects on Growth, Nutrient Accumulation, and Root Morphology of Lotus tenuis

Both saline and alkaline conditions frequently coexist in nature; however, little is known about the effects of alkaline and salt?Calkaline stresses on plants. We performed pot experiments with four treatments, control without salt addition and three stress conditions??neutral, alkaline, and mixed salt?Calkaline??to determine their effects on growth, nutrient accumulation and root architecture in the glycophytic species Lotus tenuis. Neutral and alkaline salts produced a similar detrimental effect on L. tenuis growth, whereas the effect of their combination was synergistic. Neutral salt addition, alone or mixed with NaHCO₃, led to significant leaf Na⁺ build up and reduced K⁺ concentration. In contrast, in plants treated with NaHCO₃ only, Na⁺ levels and the Na⁺/K⁺ ratio remained relatively unchanged. Proline accumulation was not affected by the high pH in the absence of NaCl, but it was raised by the neutral salt and mixed treatments. The total root length was reduced by the addition of NaCl alone, whereas it was not affected by alkalinity, regardless of the presence of NaCl. The topological trend showed that alkalinity alone or mixed with NaCl turned the root more herringbone compared with control roots, whereas no significant change in this index was observed in the treatment with the neutral salt only. The pattern of morphological changes in L. tenuis root architecture after the alkaline treatment (in the absence of NaCl) was similar to that found in the mixed salt?Calkaline treatment and different from that observed in neutral salt. A unique root morphological response to the mixed salt?Calkaline stress was the reduction in the ratio between xylem vessels and root cross-sectional areas.     

Mercury and selenium interaction in vivo: effects on thioredoxin reductase and glutathione peroxidase

Mercury compounds exert toxic effects via interaction with many vital enzymes involved in antioxidant regulation, such as selenoenzymes thioredoxin reductase (TrxR) and glutathione peroxidase (GPx). Selenium supplementation can reactivate the mercury-inhibited TrxR and recover the cell viability in vitro. To gain an insight on how selenium supplementation affects mercury toxicity in vertebrates, we investigated the effects of selenium on the mercury accumulation and TrxR and GPx activities in a fish model. Juvenile zebra-seabreams were exposed either to methylmercury (MeHg) or inorganic mercury (Hg(2+)) in the presence or absence of sodium selenite (Se) for 28 days followed by 14 days of depuration. Mercury accumulation was found to be 10-fold higher under MeHg exposure than under Hg(2+) exposure. Selenium supplementation caused a half decrease of the accumulation of MeHg but did not influence Hg(2+) accumulation. Exposure to both mercurials led to a decrease of the activity of TrxR (<50% of control) in all organs. Se supplementation coincident with Hg(2+) exposure protected the thioredoxin system in fish liver. However, supplementation of Se during the depuration phase had no effects. The activity of GPx was only affected in the brain of fishes upon the exposure to MeHg and coexposure to MeHg and Se. Selenium supplementation has a limited capacity to prevent mercury effects in brain and kidney. These results demonstrate that Se supplementation plays a protective role in a tissue-specific manner and also highlight the importance of TrxR as a main target for mercurials in vivo.     

Interaction of PABPC1 with the translation initiation complex is critical to the NMD resistance of AUG-proximal nonsense mutations

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 3'-5' linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.     

Ex vivo enrichment of circulating anti-tumor T cells from both cutaneous and ocular melanoma patients: clinical implications for adoptive cell transfer therapy

Tumor-infiltrating lymphocytes (TILs) have been successfully used for adoptive cell transfer (ACT) immunotherapy; however, due to their scarce availability, this therapy is possible for a limited fraction of cutaneous melanoma patients. We assessed whether an effective protocol for ex vivo T-cell expansion from peripheral blood mononuclear cells (PBMCs), suitable for ACT of both cutaneous and ocular melanoma patients, could be identified. PBMCs from both cutaneous and ocular melanoma patients were stimulated in vitro with autologous, irradiated melanoma cells (mixed lymphocyte tumor cell culture; MLTCs) in the presence of IL-2 and IL-15 followed by the rapid expansion protocol (REP). The functional activity of these T lymphocytes was characterized and compared with that of TILs. In addition, the immune infiltration in vivo of ocular melanoma lesions was analyzed. An efficient in vitro MLTC expansion of melanoma reactive T cells was achieved from all PBMC's samples obtained in 7 cutaneous and ocular metastatic melanoma patients. Large numbers of melanoma-specific T cells could be obtained when the REP protocol was applied to these MLTCs. Most MLTCs were enriched in non-terminally differentiated T(EM) cells homogeneously expressing co-stimulatory molecules (e.g., NKG2D, CD28, CD134, CD137). A similar pattern of anti-tumor activity, in association with a more variable expression of co-stimulatory molecules, was detected on short-term in vitro cultured TILs isolated from the same patients. In these ocular melanoma patients, we observed an immune infiltrate with suppressive characteristics and a low rate of ex vivo growing TILs (28.5% of our cases). Our MLTC protocol overcomes this limitation, allowing the isolation of T lymphocytes with effector functions even in these patients. Thus, anti-tumor circulating PBMC-derived T cells could be efficiently isolated from melanoma patients by our novel ex vivo enrichment protocol. This protocol appears suitable for ACT studies of cutaneous and ocular melanoma patients.