Cyan fluorescent protein carries a constitutive mutation that prevents its dimerization

The tendency of GFP-like fluorescent proteins to dimerize in vitro is a permanent concern as it may lead to artifacts in FRET imaging applications. However, we have found recently that CFP and YFP (the couple of GFP variants mostly used in FRET studies) show no trace of association in the cytosol of living cells up to millimolar concentrations. In this study, we investigated the oligomerization properties of purified CFP, by fluorescence anisotropy and sedimentation velocity. Surprisingly, we found that CFP has a much weaker homoaffinity than other fluorescent proteins (K(d) ≥ 3 × 10(-3) M), and that this is due to the constitutive N146I mutation, originally introduced into CFP to improve its brightness.     

Xenopus MHC class II molecules. I. Identification and structural characterization

Class II antigens from the Xenopus laevis MHC (f haplotype) were identified by using a rabbit antihuman class II beta-chain serum (anti-p29boost). This xenoantiserum inhibits bidirectional Xenopus MLR (but not PHA-stimulation), recognizes the same molecules as certain MHC-linked Xenopus alloantisera, and immunoprecipitates class II molecules from Xenopus cells consistent with the tissue distribution of mammalian class II molecules. The Xenopus class II molecules are composed of two different chains, both of which are 30 to 35kD transmembrane glycoproteins. The alpha-chains have some N-terminal sequence homology with mammalian class II alpha-chains (both I-E/DR and I-A/DC); the beta-chains are directly recognized by anti-p29boost and have a markedly increased SDS gel mobility under nonreducing conditions. During biosynthesis, they are noncovalently associated with a number of other chains, including ones at 25kD, 33kD, and 40 to 45kD. The alpha-chains bear three N-linked glycans (two Endo H insensitive in mature material) and the beta-chains bear two (one Endo H insensitive). Unlike most mammalian class II molecules, the deglycosylated beta-chains are significantly larger and more acidic than the alpha-chains.     

Autoreactive T cells in mercury-induced autoimmunity. Ability to induce the autoimmune disease

It has been previously shown that autoreactive T cells appear during mercury-induced autoimmunity in Brown-Norway (BN) rats. In the present work, it is shown that: 1) T cells and T helper cells from HgCl2-injected BN rats are able to actively transfer autoimmunity in normal BN rats; the disease transferred is exacerbated when recipients are treated with the antisuppressor/cytotoxic T cell monoclonal antibody (OX8); 2) normal T cells preincubated with HgCl2 are also able to transfer the disease in OX8-treated but not in T cell-depleted rats; and 3) T cells from HgCl2-injected BN rats also transferred the disease in both normal and T cell depleted rats. It is concluded that: 1) autoreactive T cells, and presumably anti-Ia T cells are involved in the pathogenesis of mercury-induced autoimmunity; 2) these autoreactive T cells induce suppressor/cytotoxic T cells to proliferate in normal syngeneic recipients; the fact that this T cell subset did not proliferate in HgCl2-injected BN rats suggests that HgCl2 also affects T suppressor cells; and 3) mercury-induced autoimmunity could result from the additive effect of the emergence of autoreactive T cells and of a defect at the T suppressor level.     

SIRT1 Limits Adipocyte Hyperplasia through c-Myc Inhibition

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hyperplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD⁺-dependent protein deacetylase sirtuin 1 (SIRT1), a key regulator of mammalian metabolism, maintains proper metabolic functions in many tissues, counteracting obesity. Here we report that differentiated adipocytes are hyperplastic when SIRT1 is knocked down stably in mouse 3T3-L1 preadipocytes. This phenotype is associated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveal that the c-Myc pathway is altered to drive enhanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced, and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Remarkably, differentiating SIRT1-silenced preadipocytes exhibit enhanced mitotic clonal expansion accompanied by reduced levels of p27 as well as elevated levels of CCAAT/enhancer-binding protein β (C/EBPβ) and c-Myc, which is also hyperacetylated. c-Myc activation and enhanced proliferation phenotype are also found to be SIRT1-dependent in proliferating mouse embryonic fibroblasts and differentiating human SW872 preadipocytes. Reducing both SIRT1 and c-Myc expression in 3T3-L1 cells simultaneously does not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. A better understanding of the molecular mechanisms of adipocyte hyperplasia will open new avenues toward understanding obesity.     

A major histocompatibility complex in the toadxenopus laevis (Daudin)

The genetic relationship between mixed leukocyte reaction (MLR), skin graft rejection, and some red blood cell antigens has been studied in a sibship of the toadXenopus laevis. MLR typing was achieved using blood lymphocytes. The graft experiments were performed at 17–19°C. Grafts exchanged between MLR identical sibs were rejected in 30.9±5.1 days, grafts exchanged between MLR different sibs were rejected in 20.4±2.4 days when animals differed at one MLR haplotype, and in 18.6±1.9 days when they differed at two MLR haplotypes. Immunizations and absorptions following the MLR typing produced agglutinating antisera that recognize red blood cell antigens segregating with MLR haplotypes. The results parallel those obtained in various mammalian and avian species and suggest that the homology of the major histocompatibility complex (MHC), described in higher vertebrates, can be extended to amphibians.     

Major histocompatibility complex-encoded class I molecules are absent in immunologically competent Xenopus before metamorphosis

The expression of class I and class II major histocompatibility complex (MHC)-encoded antigens has been examined at various stages of the development of the clawed frog, Xenopus. By immunoprecipitation with alloantisera or xenoantisera from radio-labeled spleen and thymus lysates, and by mixed lymphocyte reaction analysis, it was determined that the same class II molecules are expressed throughout ontogeny. In contrast, by fluorescence on frozen sections of tadpoles and by immunoprecipitation, the class I molecule is not detected in tadpoles, but appears on all tissues at the climax of metamorphosis. Animals maintained as tadpoles for long periods of time by chemical treatment do express class I antigens; thus, their expression can be independent of other biochemical and morphological changes that occur at metamorphosis. Immunofluorescence detects an otherwise uncharacterized MHC-linked alloantigen on tadpole thymic epithelium from the earliest stages of thymus differentiation.     

Cytoplasmic pH Regulation in Acer pseudoplatanus Cells: I. A P NMR Description of Acid-Load Effects

Modifications of cytoplasmic pH in Acer pseudoplatanus L. cells cultivated in suspension have been induced by acid-loads and studied by using ³¹P nuclear magnetic resonance spectroscopy. The initial drop of cytoplasmic pH, observed in the first minutes of exposure to weak lipophilic acids, was followed by a slow recovery to reach a plateau phase with a pH value lower than the initial one. Conversely, removal of the acid led to a sharp increase of cytoplasmic pH with in most cases an overshoot toward more alkaline values than the initial one and a subsequent decrease to more acidic values. This shows that A. pseudoplatanus cells powerfully regulate their cytoplasmic pH both on the acid side of their normal pH, during the acid-load, and on the alkaline side, after removal of acid. Similar results were obtained with different types of acid-loads, i.e. treatments with propionic or benzoic acid or bubbling with CO₂-enriched air. This indicates that the occurrence of pH regulation does not depend upon the method used to acid-load the cells. The time courses of cytoplasmic pH observed for A. pseudoplatanus and also Catharanthus roseus cells are similar to those recorded for animal cells but different from those described for other plant materials for which no recovery phase was observed. This can be explained by different balances between the initial rate of proton influx brought in by the acids, and the capacity of proton consumption by the regulatory mechanisms. The existence of the recovery phase offers a unique possibility to study the regulation of the cytoplasmic pH of plant cells, as it has been done in animal systems.     

Cytoplasmic pH Regulation in Acer pseudoplatanus Cells: II. Possible Mechanisms Involved in pH Regulation during Acid-Load

Qualitative and quantitative aspects of the mechanisms involved in the regulation of cytoplasmic pH during an acid-load have been studied in Acer pseudoplatanus cells. Two main processes, with about the same relative importance, account for the removal of H⁺ from the cytoplasm, namely a `metabolic consumption' of protons and the excretion of protons or proton-equivalents out of the cells. The metabolic component corresponds to a change in the equilibrium between malate synthesis and degradation leading to a 30% decrease of the malate content of the cells during the period of cytoplasmic pH regulation. Various conditions which severely inhibit the activity of the plasmalemma proton pump ATPase reduce, at most by 50%, the excretion of H⁺. This suggests that, besides the plasmalemma proton-pump, other systems are involved in the excretion of proton-equivalents. Indirect information on qualitative and quantitative features of these systems is described, which suggests the involvement of Na⁺ and HCO₃⁻ exchanges in the regulation of cytoplasmic pH of acid-loaded cells.     

The ontogeny of diversification at the immunoglobulin heavy chain locus in Xenopus.

Since the larval and adult antibody responses are distinct and restricted in the clawed toad Xenopus, it offers a near ideal model for studying the ontogeny of antibody repertoires and the mechanisms involved. Immunoglobulin heavy chain (IgH) cDNA clones and B cell IgH DNA clones from various larval and adult libraries have been analysed in isogenic Xenopus. Some features are similar in adults and tadpoles, while others differ and explain the particularities observed previously at the protein level. Among the similarities we found are: (i) the mode of rearrangements (there are approximately 50% abortive events in B cells from both stages), (ii) VH family usage (10 of 11 known VH families are expressed proportionally to the number of VH elements per family), and (iii) JH usage (of the eight to nine Xenopus JH elements, two are used in approximately 70% of the VH regions in both stages of development). We found that there is relatively higher membrane exon expression in tadpoles compared with adults; and that most of the differences come from the diversification of CDR3 through DH usage and N diversification. Unlike in mammals, Xenopus DH elements are used with a remarkable flexibility with inversion, fusions and usage in different reading frames, but tadpoles show a strong bias for the usage of only a few DH elements and of a preferred reading frame. There is N diversification, which further increases CDR3 heterogeneity, in adult Xenopus but virtually none in tadpoles. These observations can account for the fact that larval antibody responses are less heterogeneous than those of adults.     

Simultaneous high-performance liquid chromatographic determination of quinupristin, dalfopristin and their main metabolites in human plasma

Quinupristin–dalfopristin (30:70, w/w) is a new streptogramin, which has been developed for intravenous use. A specific and sensitive HPLC method was developed to measure simultaneously quinupristin (RP 57669) and dalfopristin (RP 54476) and their main metabolites in human plasma. The metabolites measured by this method were RP 69012 (glutathione-conjugated) and RPR 100391 (cysteine-conjugated) from quinupristin and RP 12536 (natural pristinamycin IIA), from dalfopristin. Solid-phase extraction with disposable cartridges was combined with reversed-phase HPLC and fluorimetric detection for RP 57669, RP 69012 and RPR 100391 and UV detection for RP 54476 and RP 12536. The method provided good recovery and low limits of quantitation (0.025 mg l⁻¹ for RP 57669, RP 54476 and RP 12536, and of 0.010 mg l⁻¹ for RP 69012 and RPR 100391). The validated range of concentrations of the method was: 0.025–5000 mg l⁻¹ for RP 57669, RP 54476 and RP 12536 and 0.010–0.750 mg l⁻¹ for RP 69012 and RPR 100391.