Ontogenesis of α2-Adrenoceptor Coupling with GTP-Binding Proteins in the Rat Telencephalon

The ontogenesis of alpha 2-adrenoceptors and GTP-binding proteins and their coupling activity were investigated in telencephalon membranes of developing rats. The manganese-induced elevation of [3H]clonidine binding was increased in an age-dependent manner but the guanosine 5'-O-(3-thio)triphosphate-induced decrease in binding did not change. The extent of the binding of [3H]clonidine at 15 nM (saturable concentration) increased in an age-dependent manner and reached the adult level at 4 days after birth. Cholera toxin and pertussis toxin catalyzed ADP-ribosylation of proteins of 46 and 41/39 kilodaltons (kDa) in solubilized cholate extracts of the membranes. The 41/39-kDa proteins ADP-ribosylated by pertussis toxin (Gi alpha + Go alpha) were increased with age and reached the adult level at day 12, whereas the 46-kDa protein (Gs alpha) reached its peak on day 12 and then decreased to the fetal level at the adult stage. The immunoblot experiments of the homogenates with antiserum (specific antibody against alpha- and beta-subunit of GTP-binding proteins) demonstrated that the 39-kDa alpha-subunit of (Go alpha) and the 36-kDa beta-subunit of GTP-binding protein (beta 36) increased with postnatal age. In contrast, 35-kDa beta-subunit (beta 35) did not change. From these results, it is suggested that the coupling activity of alpha 2-adrenoceptor with GTP-binding protein gradually develops in a manner parallel with the increase of alpha 2-adrenoceptor and pertussis toxin sensitive GTP-binding proteins, Gi, and that alpha 39 beta 36 gamma may be related to the differentiation and/or growth of nerve cells in rat telencephalon.     

Intraspecific hybridization and its bearing on chromosome evolution inVicia narbonensis (Fabaceae)

Nine accessions ofVicia narbonensis, considered to be the wild progenitor of faba bean (Vicia faba), were investigated to ascertain the nature and extent of intraspecific karyotypic polymorphism. The chromosome complements resolved into four distinct types (A, B, C, D), and the meiotic data of F₁ hybrids (A × B, B × C, A × C) revealed that alteration in chromosome morphology is the result of segmental interchanges. The interchange complexes indicate that the parents differ from each other by 1 to 2 interchanges. It is also evident that karyotype B, and not A as previously reported, is the normal karyotype of the species, and A and C are single homozygotes for unequal interchange. The comparative karyomorphology of the parents and the hybrids, and of two interchange heterozygotes of four chromosomes each in F₁ hybrids of A × C shows that the chromosomes involved in the single interchange homozygotes (A, C) are not common and the breaks in both interchanges occurred in short and long arms of the involved chromosomes. Identification of the interchanged chromosomes in the complements and the frequency of ring and chain quadrivalents in the heterozygotes enabled location of the breakpoints. The present results provide probably the first example indicating that interchange homozygosity (A) is not only firmly established but also has enabled the species to spread further by adapting to a wide range of habitats. — The genetic relationships between A and D are very different. All seven chromosome pairs in D could be distinguished from A, and for that matter, B and C as well. From the meiotic pairing properties it is also amply clear that genome D is well differentiated from A and possibly B, and C, and deserves special status.     

Isolation of human retinal genes: recoverin cDNA and gene.

A human retina cDNA library enriched for retina-specific clones was prepared by subtraction with a non-retina population of cDNA in combination with polymerase chain reaction (PCR) amplifications. A highly retina-specific cDNA clone (1190 bp) was obtained through this library encoding a 200 amino acid protein with three calcium binding sites and 87% homology to the bovine photoreceptor protein, recoverin, which has been shown to mediate the recovery of the dark current after photoactivation, and 58% homology to the calcium-binding chick cone protein, visinin. Analysis of the gene indicated a 9-10 kb single-copy gene with at least three exons and two introns. The three exons contained the entire coding sequence, and all of the calcium-binding EF-hand regions were in putative exon 1. The recoverin gene was mapped to human chromosome 17 by hybridization to a panel of human-rodent hybrid DNAs.     

Characterization of human thyrotropin receptor-related peptide-like immunoreactivity in peripheral blood of Graves' disease.

An antiserum raised against an alignment of amino acid-(32-56), termed TSHRP-1, in the extracellular domain of human thyrotropin (TSH) receptor was used to identify the TSH receptor-like substance in plasma of Graves' disease. The dilution curve of plasma TSHRP-1-like immunoreactivity was observed in a manner parallel to the standard synthetic peptide curve in radioimmunoassay, and its molecular weight estimated approximately 60 kDa. The amounts of TSHRP-1-like immunoreactivity were significantly higher in Graves' plasma than those in plasma of normal and hypothyroid patients due to Hashimoto's thyroiditis. The present results indicate that human peripheral blood possesses a soluble form of the extracellular domain of TSH receptor which may contribute to the pathophysiology of Graves' disease.     

A novel anti-HIV synthetic peptide, T-22 ([Tyr5,12,Lys7]-polyphemusin II).

Tachyplesin and polyphemusin are antimicrobial peptides recently isolated from the hemocytes of horseshoe crabs (Tachypleus tridentatus and Limulus polyphemus). We synthesized them and their analogs and examined their antiviral activity against human immunodeficiency virus (HIV) type 1 in vitro. The infection of human T cells with the virus was markedly inhibited by some of them at low concentrations. In this structure-activity study, we found that [Tyr5,12, Lys7]-polyphemusin II, which was designated as T22, had extremely high anti-HIV activity. Its 50% inhibitory concentration (EC50) was 0.008 micrograms/ml, while its 50% cytotoxic concentration (CC50) was 54 micrograms/ml and these values were comparable to those of AZT. This result indicates that T22 would be a potential candidate for the therapy of HIV infection.     

A Nonneuronal Isoform of Cell Adhesion Molecule L1: Tissue-Specific Expression and Functional Analysis

Abstract: The cell adhesion molecule L1 is a multifunctional protein in the nervous system characterizing cell adhesion, migration, and neurite outgrowth. In addition to full-length L1, we found an alternatively spliced variant lacking both the KGHHV sequence in the extracellular part and the RSLE sequence in the cytoplasmic part of L1. This L1 variant was expressed exclusively in nonneuronal cells such as Schwann cells, astrocytes, and oligodendrocytes, in contrast to the expression of the full-length L1 in neurons and cells of neuronal origin. To investigate the functions of the L1 variant, we established cell lines transfected with a cytoplasmic short L1 (L1cs) cDNA that lacks only the 12-bp segment encoding for the RSLE sequence. The promoting activities of homophilic cell adhesion, neurite outgrowth, and neuronal cell migration of L1cs-transfected cells (L4-2) were similar to those of full-length L1-transfected cells (L3-1), but the cell migratory activity of L4-2 itself was clearly lower than that of L3-1. In conclusion, the short form of L1 is a nonneuronal type, in contrast to the neuronal type of the full-length L1. Deletion of the four amino acids RSLE in the cytoplasmic region of L1 markedly reduced cell migratory activity, suggesting an importance of the RSLE sequence for the signaling events of neuronal migration mediated by L1.     

Role of integrins in differentiation of chick retinal pigmented epithelial cells in vitro

When retinal pigmented epithelial cells (PEC) of chick embryos are cultured under appropriate conditions, the phenotype changes to that of lens cells through a process known as transdifferentiation. The first half of the process, characterized by dedifferentiation of PEC, is accompanied by a marked decrease in adhesiveness of PEC to collagen type I- or type IV-coated dishes. To understand the underlying mechanisms of this change, we analyzed the expression of integrins, which are major receptors for extracellular matrix components. Northern blot analysis with cDNA probes for chicken α3, α6, α8, αv, β1 and β5 integrin mRNA showed that the genes for all these integrins are transcribed at similar levels in PEC and dedifferentiated PEC (dePEC). Further analysis of β1 integrin, which is a major component of integrin heterodimers, showed that although the protein amount of β1 integrin was not changed, its localization at focal contacts seen in PEC was lost in dePEC. When anti-β1 integrin antibody was added to the PEC culture medium, a decrease of cell-substrate adhesiveness occurred, followed by a gradual change in both morphology and gene expression patterns to ones similar to those of dePEC. These findings suggest that an appropriate distribution of β1 integrin plays an essential role in maintaining the differentiated state of PEC through cell-substrate adhesion.