Ca2+-Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ-Imidoguanosine 5'-Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum

Abstract: With [³H]guanosine triphosphate ([³H]GTP) and [³H]β, γ -imidoguanosine 5′-triphosphate ([³H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (K_D= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [³H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [³H]GTP and [³H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [³H]GTP (into [³H]guanosine) and the likely formation of Ca-guanine nucleotide^2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [³H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [³H]GTP and [³H]GppNHp catabolism (into [³H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [³H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [³H]5-hydroxytryptamine ([³H]5-HT) in the rat brain.     

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.     

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.     

Two ancient allelic lineages at the single classical class I locus in the Xenopus MHC

Unlike all other vertebrates examined to date, there is only one detectable class I locus in the Xenopus MHC. On the bases of a nearly ubiquitous and high tissue expression, extensive polymorphism, and MHC linkage, this gene is of the classical or class Ia type. Sequencing analysis of class Ia cDNAs encoded by eight defined MHC haplotypes reveals two very old allelic lineages that perhaps emerged when humans and mice diverged from a common ancestor up to 100 million years ago. The unprecedented age of these lineages suggests that different class Ia genes from ancestors of the laboratory model Xenopus laevis are now expressed as alleles in this species. The lineages are best defined by their cytoplasmic and alpha2 peptide-binding domains, and there are highly diverse alleles (defined by the alpha1 peptide-binding domain) in each lineage. Surprisingly, the alpha3 domains are homogenized in both lineages, suggesting that interallelic gene conversion/recombination maintains the high sequence similarity.     

Genomic analysis of immunity in a Urochordate and the emergence of the vertebrate immune system: “waiting for Godot”

Genome-wide sequence analysis in the invertebrate chordate, Ciona intestinalis, has provided a comprehensive picture of immune-related genes in an organism that occupies a key phylogenetic position in vertebrate evolution. The pivotal genes for adaptive immunity, such as the major histocompatibility complex (MHC) class I and II genes, T-cell receptors, or dimeric immunoglobulin molecules, have not been identified in the Ciona genome. Many genes involved in innate immunity have been identified, including complement components, Toll-like receptors, and the genes involved in intracellular signal transduction of immune responses, and show both expansion and unexpected diversity in comparison with the vertebrates. In addition, a number of genes were identified which predicted integral membrane proteins with extracellular C-type lectin or immunoglobulin domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activation motifs (ITAMs) (plus their associated signal transduction molecules), suggesting that activating and inhibitory receptors have an MHC-independent function and an early evolutionary origin. A crucial component of vertebrate adaptive immunity is somatic diversification, and the recombination activating genes (RAG) and activation-induced cytidine deaminase (AID) genes responsible for the Generation of diversity are not present in Ciona. However, there are key V regions, the essential feature of an immunoglobulin superfamily VC1-like core, and possible proto-MHC regions scattered throughout the genome waiting for Godot.     

Influence of insulin treatment and feed restriction on follicular development in cyclic gilts

Crossbred gilts were used to investigate whether exogenous insulin can restore normal follicular growth in feed-restricted gilts. After an 18-day altrenogest treatment, the first day of oestrous behaviour was designed as day 0. From day 0 to 13, all gilts received the same amount of feed, calculated to meet 200% of the energy requirements for maintenance. On day 14, luteolysis was induced by injection of an analogue of prostaglandin F2alpha. All gilts were slaughtered on day 19 and their ovaries removed. In Experiment 1, gilts received a high (240% of maintenance) or low (80%) level of feeding (n=10/group) from day 14 to 18. The number of large follicles (> or = 5 mm) on day 19 was reduced in feed-restricted gilts (16.9 versus 20.6, P<0.05). The same protocol of feed restriction was used in Experiment 2 (240% versus 80% of maintenance from day 14 to 18), and some gilts received daily injections of insulin (0.6 IU live weight kg(-1)). The three experimental groups were H: 240% and no insulin (n=8); H-I: 240%+insulin (n=8) and L-I: 80%+insulin (n=7). On day 18, 4 h after insulin injection, plasma insulin was higher in insulin-treated than in untreated gilts and glucose concentrations were reduced more dramatically in L-I than in H-I gilts (P<0.05). Concentrations of IGF-I were lower in L-I than in other gilts (P<0.05) and plasma IGFBPs were not significantly affected by treatments. On day 19, the number of large follicles (> or = 5 mm) was not significantly influenced by treatments (19.4, 17.6 and 15.3 for H, H-I and L-I gilts, respectively). Insulin, IGF-I and IGFBP-2 levels in follicular fluids from large follicles did not differ between females whereas IGFBP-3 levels were lower in L-I than in H gilts (P<0.05) and intermediate in H-I gilts. Intrafollicular levels of glucose were higher in feed-restricted than in well-fed gilts (P<0.05). These results suggest that exogenous insulin does not restore final follicular growth impaired by acute undernutrition.     

CoPreTHi: a Web tool which combines transmembrane protein segment prediction methods

CoPreTHi is a Java based web application, which combines the results of methods that predict the location of transmembrane segments in protein sequences into a joint prediction histogram. Clearly, the joint prediction algorithm, produces superior quality results than individual prediction schemes. The program is available at http://o2.db.uoa.gr/CoPreTHi.