Halobacterial megaplasmids are negatively supercoiled

Several covalently closed circular halobacterial megaplasmids (up to more than 500 kb) from different strains of Halolerax mediterranei, have been resolved by orthogonal-field alternating gel electro-phoresis (OFAGE). These molecules seem to be negatively supercoiled in vivo, as deduced from the effect of intercalating agents affecting their topology and, therefore, their electrophoretic mobility. It has also been demonstrated that the topolsomerase II Inhibitor novobiocin affects the native topological state of halobacterial megaplasmids impeding their migration in OFAGE under standard conditions for resolution of large supercoiled molecules.     

Sheep liver beta-N-acetylhexosaminidase: partial purification, characterization and photodynamic inactivation

Sheep liver beta-N-acetylhexosaminidase was purified over 20-fold by conventional methods. The enzyme possessed activity against both p-nitrophenly-beta-D-N-acetylglucosaminide and p-nitrophenyl-beta-D-N-acetylgalactosaminide as substrates. On the basis of a variety of physical and chemical analyses including pH stability, substrate inhibition studies and photodynamic inactivation, it was concluded that both the beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities reside within the same molecule.     

Function in the Immune System of Antigenic Peptides from S-Antigen (Arrestin)

A significant amount of information concerning immunologic domains of an antigenic molecule can be obtained by studying its peptides. We describe a method for identifying and characterizing immunologically relevant T-cell and B-cell epitopes in S-antigen, a well-characterized, highly pathogenic retinal autoantigen for the induction of experimental autoimmune uveitis. The method involves the generation of peptide fragments by enzymatic treatment of native S-antigen and by the simultaneous synthesis of large numbers of peptides in small quantities for screening and testing. Peptides demonstrating T- or B-cell activity are then synthesized in large quantity for additional studies. Although useful information was obtained by the use of enzymatically generated peptides, synthetic peptides provided the greatest flexibility and specificity, allowing the precise localization of amino acid sequences of S-antigen required for a particular immunological function such as antibody binding, T-cell proliferative responses, pathogenicity, and the induction of tolerance. These studies have wide applicability to the study of other antigenic molecules and have led to a better understanding of the immune mechanisms involved in the pathogenesis of experimental autoimmune uveitis. This, in turn, provides a basis for the processes that may be occurring in certain forms of human uveitis.     

Molecular mimicry between a uveitopathogenic site of S-antigen and viral peptides. Induction of experimental autoimmune uveitis in Lewis rats.

S-Antigen (S-Ag) is a well characterized 45,000 m.w. photoreceptor cell protein. When injected into susceptible animal species, including primates, it induces an experimental autoimmune uveitis, a predominantly T cell-mediated autoimmune disease of the retina and uveal tract of the eye, and of the pineal gland. In this study we found an amino acid sequence homology between a uveitopathogenic site of S-Ag, several viral proteins and one additional nonviral protein. An experimental autoimmune uveitis and pinealitis was induced in Lewis rats with these different synthetic peptides, corresponding to the amino sequence of hepatitis B virus DNA polymerase, gag-pol polyprotein of Baboon endogenous virus and gag-pol polyprotein of AKV murine leukemia virus and potato proteinase inhibitor IIa, which contain three or more consecutive amino acids identical to peptide M in S-Ag. Lymph node cells from rats immunized with either peptide M or the different synthetic peptides showed a significant degree of cross-reaction. Mononuclear cells from monkeys (Macaca fascicularis) immunized with peptide M also showed significant proliferation when incubated with either peptide M or synthetic peptides as measured by in vitro lymphocyte mitogenesis assay using [3H]TdR. Based on our findings we conclude that a viral infection may sensitize the mononuclear cells that can cross-react with self proteins by a mechanism termed molecular mimicry. Tissue injury from the resultant autoantigenic event can take place in the absence of the infectious virus that initiated the immune response.     

Changes in the cellular permeability of the embryonic axis inGcer arietinum L. seeds during germination

The effect of short chain fatty acids on the cellular permeability of embryonic axis inGcer arietinum seeds was studied. Octanoic (OCT) and nonanoic (NON) adds, which reduce both germination and growth of the embryonic axis and raise the inhibitor effects of the supraoptimal temperatures (30?C), induce a greater ionic efflux into the medium (conductivity). NON reduces glucose (3-0-MG) and K⁺ (⁸⁶Rb) uptake during the germinative process, this action being counteracted by fusicoccin (FC) at optimal (25?C) and supraoptimal temperatures (30 ?C). Tonoplast and plasmalemma increase their permeability to the K⁺ efflux when NON is present. Addition of NON+FC gives rise to higher values in the efflux rate, the vacuolar compartment being the most affected. Temperatures around zero (2 ?C) notably reduce the isotope efflux from cytosol and vacuole. NON acid does not significantly affect the efflux of³H-ABA, suggesting that it does not cause any important changes in the phytohormone compartmentation.     

Sequence homology between yeast histone H3 and uveitopathogenic site of S-antigen: lymphocyte cross-reaction and adoptive transfer of the disease

Experimental autoimmune uveitis (EAU) serves as an animal model of ocular inflammation. The disease is caused by the immunization of microgram amounts of a soluble retinal protein, designated S-antigen, in susceptible animal strains, including primates. We induced EAU and experimental autoimmune pinealitis (EAP) in Lewis rats with a small synthetic peptide corresponding to amino acid positions 106-121 in yeast histone H3. This peptide contains five consecutive amino acids identical to a uveitopathogenic site (peptide M) in human S-antigen. Lymph node or mononuclear cells from different species of animals immunized either with histone H3 or with peptide M showed significant cross-reaction as measured by in vitro lymphocyte mitogenesis assay using [3H]thymidine. Also, we adoptively transferred the EAU and EAP in naive rats by immune lymph node cells. These findings support the fact that selected bacterial, viral, or fungal proteins with amino acid sequence homologies to normal retinal proteins are uveitopathogenic and, as such, provide a basis for autoimmune inflammatory diseases.     

Rat pineal S-antigen: sequence analysis reveals presence of alpha-transducin homologous sequence

S-antigen (S-Ag) is a soluble, highly antigenic protein, the administration of which induces autoimmune uveitis. This protein is found in the retina and pineal. Retinal S-Ag from three species has been sequenced. In this study rat pineal S-Ag was sequenced. Clones were isolated from a rat pineal lambda gt11 cDNA library by probing with a 300 bp fragment of mouse retinal S-Ag cDNA containing the 5'-coding region. The largest clone isolated (RPS-118; 1364 bp) contained the entire coding sequence. Comparison of the rat pineal and mouse retinal S-Ag nucleotide sequences indicated a high homology (95%). The deduced amino acid sequence was found to contain 403 residues (congruent to 44 992 Da). Comparison of the rat pineal and mouse retinal S-Ag amino acid sequences also revealed high homology (97%). The similarity of both the nucleotide and amino acid sequences of rat pineal and mouse retinal S-Ag indicates that expression of the S-Ag gene in both tissues is similar. Further analysis of the rat pineal S-Ag sequence indicated that it contained essentially the same major uveitopathogenic region of S-Ag present in bovine retina; minor uveitopathogenic sites were somewhat different. As is true of retinal S-Ag, rat pineal S-Ag contains the same consensus phosphoryl-binding site present in many GTP/GDP-binding proteins and a homologous sequence found in the C-terminus of alpha-transducin. These sequences may play a role in the action of pineal S-Ag in transmembrane signal transduction.     

Human esterase D gene: complete cDNA sequence,genomic structure,and application in the genetic diagnosis of human retinoblastoma

Summary The gene encoding human esterase D (EsD), a member of the nonspecific esterase family, is a useful genetic marker for retinoblastoma (RB) and Wilson's disease. Previously we identified a cDNA clone from this gene and determined its chromosomal location. In this report, we present the complete cDNA sequence of the human EsD gene. A long open reading frame encoded a predicted protein of 282 amino acids with molecular weight of 30 kD. A computer-assisted search of a protein sequence data base revealed homology with two other esterases, acetylcholinesterase of Torpedo and esterase-6 of Drosophila. Homologous region were centered around presumptive active sites, suggesting that the catalytic domains of the esterases are conserved during evolution. Three genomic clones of this gene were also isolated and characterized by restriction mapping. At least ten exons were distributed over a 35-kb (kilobase pair) region; each exon contained an average of 100 basepairs (bp). A polymorphic site for Apa I, located within an intron of the esterase D gene, can be used to identify chromosome 13 carrying defective RB alleles within retinoblastoma families.     

The distribution of A1 adenosine receptor and 5′-nucleotidase in pig brain cortex subcellular fractions

Pig brain cerebral cortex was subfractionated by isopycnic centrifugation in sucrose gradients. In each subfraction the content of the agonist [³H]R-PIA binding, the activity of adenosine metabolizing enzymes (5-nucleotidase and adenosine deaminase) and the activity of membrane marker enzymes were determined. The fractions were also examined by electron microscope. In general, the results suggest a widespread distribution of A₁ adenosine receptors in membranes from different origins. Marker enzyme profile characterization indicated an enrichment of A₁ adenosine receptor in pre-synaptic membranes isolated from the crude synaptosomal fraction (P2B subfraction) as well as in membranes of glial origin such as myelin. The receptor is also present in the endoplasmic reticulum and in membranes isolated from the microsomal fraction that seem to have a post-synaptic origin (P3B). In subfractions having a high content of adenosine receptor the equilibrium binding paramters were obtained as well as the proportion of high- to low-affinity sites. From the values of the equilibrium constants it was not possible to find differences between the receptor in the different subfractions. Analysis of the affinity state distribution showed a diminished percentage of high-affinity sites in fraction P3A, which can be accounted by the existence of myelin membranes; in contrast the percentage of high-affinity states was higher in P2 and P3B, indicating that in these fractions the receptor is present in synaptosomal membranes. The close correlation shown between the enzyme 5-nucleotidase specific activity and the specific ligand binding distributions led us to postulate an important role for the enzyme in the regulation of adenosine action in pig brain cortex.