Immunological analysis of hemoglobin transition during metamorphosis of normal and isogenicXenopus

Summary Antisera against larval and adultXenopus hemoglobins as well as adult human hemoglobin showed no cross-reaction when tested by immunodiffusion against each heterologous antigen. In this test hemoglobin of a single animal produced two precipitation lines for larvae, but only one for adult stages. Immunoelectrophoresis also revealed more complex precipitation patterns for larval than for adult hemoglobins. Hemoglobin of the isogenic hybrid cloneXenopus laevis/X. gilli also reacted with antisera against normalXenopus hemoglobin.Quantitation of hemoglobins, analyzed by radial immunodiffusion showed fewer than 1% of adult hemoglobin in red cells of larvae, but 30% at completion of metamorphosis. Two weeks later adult hemoglobin attained over 90%, and in red cells of adultXenopus an average of 1% larval hemoglobin were detected.The relatively short transition period suggests that the loss of larval hemoglobin may be due to the elimination of larval red cells, and that the increase in adult hemoglobin may be indicative of a new cell line.     

Radiobiological evaluation of the suitability of negative pions in tumor therapy; facts and theories

Summary A successful cancer radiotherapy consists mainly in sparing normal tissues and killing malignant cells. Negative pions seem to have several and unique gain factors, as Fowler and Perkins 1961 proposed. With the biomedical pion channel of the 590 MeV proton-accelerator of the Swiss Institute for Nuclear Research (SIN) we had since several years the opportunity to test some theoretical conceptions in comparison to several preliminary experiments performed with pions of low dose rate (Berkeley, CERN, Nimrod). The dosimetric measurements showed for the momentum of 180 MeV/c and a ratio e^–/ ^– of 0.1 an excellent depth curve with p. ex: a peak/plateau ratio of 2.5. Besides this important gain factor for radiotherapy, negative pions have the unique particularity to act on the tissues in the same treatment with two different types of radiations, in the peak (treatment volume) with high LET radiation (presumably high RBE) and low LET radiation in the plateau. Following systems have been used: Inactivation of single mammalian cells, induction of chromatid aberrations in Chinese hamster cells; small intestine of mouse (early and late effects); early and late effects in mouse foot; induction of anomalies in mouse embryos; induction of cerebral microvascular damages in neonatal rats; proliferation of Ehrlichascites carcinoma cells; induction of different types of mutation in different stages of male germ cells and somatic cells (Drosophila). The RBE-values in the peak region vary between 0.7–3.3, and are different even in the same system with the same end point but at different cell stages and conditions. For the plateau region the RBE-values lie mostly under 1 (compared with 140 kV-photons) and can be identical with 29 MeV-photons. The clinically important peak/plateau relation lies in every experiment over 1 and reaches even the value of 4.2. The unexpected RBE-values in peak under 1 lead to a new conception of RBE, the two system theory. In intrinsic radiosensitive euoxic systems (healthy tissue) the RBE of peak (star) pions can be under 1, in intrinsic radioresistant hypoxic systems (tumor cells) in contrary over 1. The two systems can also have different vulnerability of repair svstem.Invited paper, presented at the 14th Annual Meeting of European Society of Radiation Biology, Jülich, Germany, October 8–14, 1978Supported by the Swiss National Science Foundation (grant no. 3.682-0.75)Prof. Dr. A. Prader dedicated on the occasion of his 60th birthday     

Incorporating fluorescent dyes and quantum dots into magnetic microbeads for immunoassays

Microbeads that are both paramagnetic and fluorescently labeled are commercially available in colors spanning the visible spectrum. Although these commercial beads can be bright, polydispersity in both size and fluorescent intensity limit their use in quantitative assays. Very recently, more monodisperse beads have become available, but their large size and surface properties make them less than ideal for some bioassay applications. Here we describe methods to customize commercial nonfluorescent magnetic microparticles with fluorescent dyes and quantum dots (QDs) without affecting their magnetic or surface chemical properties. Fluorescent dyes and 3.3-nm diameter CdSe/ZnS QDs were sequestered within 0.8-micron diameter magnetic beads by swelling the polystyrene matrix of the bead in organic solvent, letting the chromophores partition, and then collapsing the matrix in polar solvents. Chromophore incorporation has been characterized using both UV-visible absorption spectroscopy and fluorescence microscopy, with an average of 3 x 10(8) rhodamine 6G molecules/bead and 6 x 10(4) QDs/bead. The modified beads are uniform in size and intensity, with optical properties comparable to currently available commercial beads. Immunoassay results obtained with our custom fluorescent magnetic microbeads are consistent with those obtained using conventional magnetic microbeads.     

Automatic target selection for structural genomics on eukaryotes

A central goal of structural genomics is to experimentally determine representative structures for all protein families. At least 14 structural genomics pilot projects are currently investigating the feasibility of high-throughput structure determination; the National Institutes of Health funded nine of these in the United States. Initiatives differ in the particular subset of "all families" on which they focus. At the NorthEast Structural Genomics consortium (NESG), we target eukaryotic protein domain families. The automatic target selection procedure has three aims: 1) identify all protein domain families from currently five entirely sequenced eukaryotic target organisms based on their sequence homology, 2) discard those families that can be modeled on the basis of structural information already present in the PDB, and 3) target representatives of the remaining families for structure determination. To guarantee that all members of one family share a common foldlike region, we had to begin by dissecting proteins into structural domain-like regions before clustering. Our hierarchical approach, CHOP, utilizing homology to PrISM, Pfam-A, and SWISS-PROT chopped the 103,796 eukaryotic proteins/ORFs into 247,222 fragments. Of these fragments, 122,999 appeared suitable targets that were grouped into >27,000 singletons and >18,000 multifragment clusters. Thus, our results suggested that it might be necessary to determine >40,000 structures to minimally cover the subset of five eukaryotic proteomes.     

Potentiation of a tumor cell susceptibility to autologous CTL killing by restoration of wild-type p53 function

Inactivation of p53 has been implicated in many types of tumors particularly in non-small cell lung carcinoma, one of the most common cancers in which p53 mutation has been frequently identified. The aim of this study was to investigate the influence of p53 status on the regulation of tumor susceptibility to specific CTL-mediated cell death. For this purpose, we used a cytotoxic T lymphocyte clone, Heu127, able to lyse the human autologous lung carcinoma cell line, IGR-Heu, in a HLA-A2-restricted manner. Direct genomic DNA sequencing revealed that IGR-Heu expresses a mutated p53 at codon 132 of the exon 5 which results in the loss of p53 capacity to induce the expression of the p53-regulated gene product p21(waf/CIP1). Initial experiments demonstrated that IGR-Heu was resistant to Fas, TNF, and TRAIL apoptotic pathways. This correlated with the lack of p55 TNFRI, Fas, DR4, and DR5 expression. The effect of wild-type (wt) p53 restoration on the sensitization of IGR-Heu to autologous CTL clone lysis was investigated following infection of the tumor cell line with a recombinant adenovirus encoding the wt p53 (Adwtp53). We demonstrate that the restoration of wt p53 expression and function resulted in a significant potentiation of target cell susceptibility to CTL-mediated lysis. The wt p53-induced optimization of tumor cell killing by specific CTL involves at least in part Fas-mediated pathway via induction of CD95 expression by tumor cells but does not appear to interfere with granzyme B cytotoxic pathway.     

Lake Baikal climatic record between 310 and 50 ky BP: Interplay between diatoms, watershed weathering and orbital forcing

The environmental record from Lake Baikal, Russia, from 310 to 50 ky BP (MIS 9a to MIS 3) was interpreted using rock magnetic, UV–Vis spectral, mineralogical, and diatom analyses. The age model was based on a correlation of the diatom and chemical weathering records and the summer insolation curve at 55°N and checked against an age model based on the proxy of relative palaeointensity of the Earth's magnetic field. Peaks in chemical weathering within the watershed, inferred from maximum concentration of magnetic and coloured minerals and mica, the lowest mean Fe oxidation state in silicates and highs in expandable clay minerals correlated with the Northern Hemisphere summer insolation minima at 55°N. Reconstructed changes in weathering intensity are better correlated to insolation patterns than to global ice volume records. We propose a scheme of yet missing palaeoenvironmental interpretation of the diatom assemblage, including also some extinct species. Aulacoseira baicalensis and Aulacoseira skvortzowii were abundant in the early stages of lake flora recovery immediately after deglaciation and during MIS 7e and MIS 5e; periods of more pronounced continental climate and peak chemical weathering. Stephanodiscus formosus var. minor, Cyclotella minuta and Cyclotella ornata dominated in intervals of decreased seasonality and decreased humidity at the end of most interglacial/interstadial diatom zones. Stephanodiscus grandis, Stephanodiscus carconeiformis and Stephanodiscus formosus were ubiquitous between MIS 8 and MIS 5, an interval marked by high seasonality, i.e., large differences between winter and summer insolation, and low humidity revealed by a low hydrolysis of expandable clay minerals in the watershed. Diatom concentrations peaked in the climatic optima of MIS 7e and MIS 5e and in the short periods marked by shifts to warmer conditions in the upper sections of MIS 5: MIS 5c (103–99 ky BP), MIS 5b (90–88 ky BP), and MIS 5a (84–79 ky BP) in which increased humidity resulted in enhanced hydrolysis of clay minerals. No such short similar climatic optimums were found from MIS 9a to MIS 6. Sharp climate deteriorations recorded in the diatom and clay mineral records at 107, 94, and 87 ky BP, however, occurred within 1–2 ky of cold extremes in North Atlantic sea surface temperature emphasizing the strong teleconnections between the two localities.     

Polymorphisms in the RANTES gene increase susceptibility to active tuberculosis in Tunisia

RANTES plays a pivotal role in attracting and activating various leukocyte populations that control Mycobacterium tuberculosis infection. The present study investigated the relationship between the RANTES polymorphisms (-28C/G; rs2280788, and -403G/A; rs2107538) and susceptibility to active tuberculosis (TB) in Tunisian populations. A total of 168 patients with pulmonary TB (pTB), 55 with extrapulmonary TB (epTB), and 150 control subjects were studied. Genotype analyses were carried out using polymerase chain reaction-restriction fragment length polymorphism method. We found that the -28 GG genotype was significantly associated with susceptibility to pTB (odds ratio [OR]=11.19; 95% confidence intervals [CI], 5.14-25; P corrected for the number of genotypes [Pc]=10(-8)) and epTB (OR=11.67; 95% CI, 4.74-29.33; Pc=10(-8)). However, the -28 CC genotype was found to be significantly associated with resistance to pTB (OR=0.08; 95% CI, 0.04-0.16; Pc=10(-8)) and epTB development (OR=0.11; 95% CI, 0.05-0.27; Pc=10(-8)). -403A allele was associated with increased risk development of epTB (OR=2.21; 95% CI, 1.18-4.14; p=0.007). G-G and A-C haplotypes and the AG/GC diplotype were associated with increase susceptibility to pTB (OR=7.88, 95% CI, 5.38-11.55; Pc=3.10(-8); OR=2.32, 95% CI, 1.32-4.11; Pc=3.10(-3); OR=13.26, 95% CI, 6.06-29.89; Pc=3.10(-8); respectively) and epTB (OR=6.64, 95% CI, 4-11.05; Pc=3.10(-8); OR=2.6, 95% CI, 1.26-5.35; Pc=12.10(-3); OR=11.26, 95% CI, 4.44-29.28; Pc=3.10(-8); respectively). Collectively, our findings suggested an association of the RANTES -28C/G and -403G/A functional polymorphisms with susceptibility to Mycobacterium tuberculosis infection in Tunisian populations.     

Inhibition of IL-6, TNF-alpha, and cyclooxygenase-2 protein expression by prostaglandin E2-induced IL-10 in bone marrow-derived dendritic cells

Several endogenously produced mediators, including cytokines such as IL-6, IL-10, and TNF-alpha and prostanoids such as prostaglandin E(2) (PGE(2)), regulate dendritic cell (DC) function and contribute to immune homeostasis. In this study, we report that exogenous PGE(2) enhances the production of IL-10 from bone marrow-derived DC (BM-DC). IL-6, but not TNF-alpha, release is enhanced by PGE(2) in the presence of anti-IL-10, suggesting that endogenous IL-10 masks PGE(2)-induced IL-6. Furthermore, both exogenous IL-10 and PGE(2) inhibit LPS-induced IL-6 and TNF-alpha, whereas selective inhibition of cyclooxygenase-2 (COX-2) or addition of anti-IL-10 causes the reverse effects. Exogenous IL-10, but not IL-6, dose-dependently suppresses COX-2 protein expression and PGE(2) production, and TNF-alpha does not reverse this effect. In contrast, anti-IL-10 up-regulates prostanoid production by LPS-stimulated BM-DC. Taken together, our results show that in response to PGE(2), BM-DC produce IL-10, which in turn down-regulates their own production of IL-6-, TNF-alpha-, and COX-2-derived prostanoids, and plays crucial roles in determining the BM-DC pro-inflammatory phenotype.