Eradication of metastatic tumour cells from lymph nodes by local administration of anti-CD3 antibody

The possibility of in vivo removal of metastatic tumour cells from lymph nodes by local intradermal administration of an anti-CD3 monoclonal antibody (mAb) was examined. Murine tumour cells in the lymph nodes were completely eradicated by intradermal injections of the mAb. This treatment was effective for removal of Lewis lung cancer cells from lymph nodes, but not for removal of subcutaneous tumours of this cell line. This treatment induced in vivo cytotoxicity in the regional lymph nodes against the syngeneic tumour cells. The following in vitro studies suggested that the cytotoxicity was probably mediated mainly by CD4⁺ T cells, with slight participation of CD8⁺ T cells. Normal lymph node and spleen cells showed cytotoxicity after in vitro incubation with the mAb for 2 days. Cell sorting with a fluoresceinactivated cell sorter showed that CD4⁺ T cells developed during the incubation to lyse syngeneic tumor cells directly by themselves, macrophages not being involved in this tumour cell lysis. The lytic activity was detected in the cellular fractions, but not in the culture supernatants of these T cells. Furthermore, it was completely blocked by specific antiserum for tumour necrosis factor- (TNF). An immunoprecipitation study revealed that these T cells expressed TNF molecules of 26 kDa, but not of 17 kDa, suggesting that tumour cell lysis was caused by membraneintegrated TNF molecules. These results strongly suggest that local administration of anti-CD3 antibody is a very effective and appropriate procedure for eradication of metastatic tumour cells from regional lymph nodes.     

Identification and biological activity of novel peptidomimetic gastrin/CCK-B receptor agonists

The design, synthesis and biological activity of two novel series of compounds derived from the basic Boc-CCK-4 structure which provide potent ligands for the gastrin/CCK-B receptor is outlined. Within these series, new pseudopeptide compounds were discovered which unexpectedly were functional agonists in vivo, as shown by their ability to stimulate basal gastric acid secretion in rats, an effect which was blocked by the potent gastrin/CCK-B receptor antagonist YM022.     

Induction of sexual co-flocculation of heterothallic fission yeast (Schizosaccharomyces pombe) cells by mating pheromones

Heterothallic fission yeast (Schizosaccharomyces pombe) cells preincubated with sex pheromone, P- or M-factor of the obverse mating-type cells, in mannose synthetic medium (MSM) results in remarkably increased sexual co-flocculation with obverse mating-type cells almost without time lag, i.e., within 10 min. By contrast, comparable flocculation requires over 1 h if untreated control cells are mixed with obverse mating-type cells. The agglutinin of P cells is more inducible than that of M cells. These pheromonal inductions of sexual co-flocculation are inhibited by the addition of cycloheximide or tunicamycin during preincubation but not by chloramphenicol or hydroxyurea. These results demonstrate that, in addition to (a) the repression of cell division (G1 arrest) and (b) the activation of cell wall autolytic processes (mating-specific elongation of cells: formation of their conjugation tubes), mating pheromones of fission yeast have another important role; (c) to induce sexual co-flocculation (agglutinability). Using our experimental system of preincubation with sexual pheromones, we show that M-agglutinin is heat-stable and its induction is inhibited by tunicamycin, but that P-agglutinin is heat-labile and its induction is only partially inhibited by tunicamycin.     

Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope.

Unilamellar liposomes with diameters of 25-100 microns were prepared in various physiological salt solutions, e.g., 100 mM KCl plus 1 mM CaCl2. Successful preparation of the giant liposomes at high ionic strengths required the inclusion of 10-20% of a charged lipid, such as phosphatidylglycerol, phosphatidylserine, phosphatidic acid, or cardiolipin, in phosphatidylcholine or phosphatidylethanolamine. Three criteria were employed to identify unilamellar liposomes, yielding consistent results. Under a phase-contrast microscope those liposomes that showed the thinnest contour and had a vigorously undulating membrane were judged unilamellar. When liposomes were stained with the lipophilic fluorescent dye octadecyl rhodamine B, fluorescence intensities of the membrane of individual liposomes were integer multiples (up to four) of the lowest ones, the least fluorescent liposomes being those also judged unilamellar in the phase-contrast image. Micropipette aspiration test showed that the liposomes judged unilamellar in phase and fluorescence images had an area elastic modulus of approximately 160 dyn/cm, in agreement with literature values. The giant liposomes were stable and retained a concentration gradient of K+ across the membrane, as evidenced in fluorescence images of the K(+)-indicator PBFI encapsulated in the liposomes. Ionophore-induced K+ transport and associated volume change were observed in individual liposomes.     

Genetically engineered antibodies which have reduced binding affinity for thyroxine but retain the same affinity for tri-iodothyronine

Summary Based on a three-dimensional molecular model of the variable region of a monoclonal antibody (Ab) TT1, in which the complementarity determining regions (CDRs) associate to form a cavity large enough to accommodate a single molecule of tri-iodothyronine (T3) orthyroxine (T4), we designed TT1 mutants with one amino acid substitution as candidates which have their binding affinity for T4 reduced but retain the same affinity for T3. Each candidate was subsequently tested by site-directed mutagenesis, transient expression in COS cells, and surface plasmon resonance (SPR) analysis for its binding ability for T3- or T4-conjugates with alkaline phosphatase (AP). Of the candidates, the Ab with serine in place of glycine at position 92 of the light chain (L;G92S) and the Ab with alanine in place of leucine at position 47 of the heavy chain (H;L47A) had the association constant (KA = kass / kdiss) for binding to T4-AP decreased by 5-fold, but retained the same KA for T3-AP.     

Distribution and characterization of diazepam binding inhibitor (DBI) in peripheral tissues of rat

We studied the expression and distribution of the polypeptide diazepam binding inhibitor (DBI) in rat peripheral organs by immunocytochemistry, radioimmunoassay, Northern blot analysis and binding assay. Variable amounts of the DBI peptide and DBI mRNA were found in all the tissues examined (liver, duodenum, testis, kidney, adrenal gland, heart, ovary, lung, skeletal muscle and spleen), with the highest level of expression in liver (220 pmol of DBI/mg protein) and the lowest in spleen (11 pmol of DBI/mg protein). A good correlation between DBI-like immunoreactivity (DBI-LI) and mRNA content was found in all tissues except the heart. The immunohistochemical analysis revealed discrete localization of DBI-LI in cell types with specialized functions: for example, the highest DBI-LI content was found in steroid-producing cells (glomerulosa and fasciculata cells of adrenal cortex, Leydig cells of testis); lower DBI-LI immunostaining was found in epithelial cells specialized for water and electrolyte transport (intestinal mucosa, distal convoluted tubules of kidney). Hepatic cells contained moderate immunoreactivity however the total content of DBI in liver is relatively high and is due to the diffuse presence of DBI in every hepatocyte. Cells with high expression of DBI have been shown to contain a high density of mitochondrial benzodiazepine (BZ) binding sites. This observation led us to perform a competitive binding assay between DBI and [3H]PK11195 (a ligand for the mitochondrial BZ binding sites) on mitochondrial membranes of adrenal cortical cells. In this experiment, DBI yielded an apparent competitive inhibition of the binding of PK11195 to the BZ binding sites. Our data support a possible role for DBI as endogenous regulator of intracellular metabolic functions, such as steroidogenesis, via the mitochondrial BZ receptors.     

Evolution of RNA polymerases and branching patterns of the three major groups of archaebacteria

Summary The amino acid sequences of the largest subunits of the RNA polymerases I, II, and III from eukaryotes were compared with those of archaebacterial and eubacterial homologs, and their evolutionary relationships were analyzed in detail by a recently developed tree-making method, the likelihood method of protein phylogeny, as well as by the neighbor-joining method and the parsimony method, together with bootstrap analyses. It was shown that the best tree topologies predicted by the first two methods are identical, whereas the last one predicts a distinct tree. The maximum likelihood tree revealed that, after the separation from archaebacteria, the three eukaryotic RNA polymerases diverged from an ancestral precursor in the eukaryotic lineage. This result is contrasted with the published result showing multiple origins for the three eukaryotic polymerases. It was shown that eukaryotic RNA polymerase I evolved much more rapidly than RNA polymerases II and III: The N-terminal half of RNA polymerase I shows an extraordinarily high evolutionary rate, possibly due to relaxed functional constraints. In contrast the evolutionary rate of archaebacterial RNA polymerase is remarkably limited. In addition, including the second largest subunit of the RNA polymerase, a detailed analysis for the branching pattern of the three major groups of archaebacteria was carried out by the maximum likelihood method. It was shown that the three major groups of archaebacteria are likely to form a single cluster; that is, archaebacteria are likely to be monophyletic as originally proposed by Woese and his colleagues.     

A novel peptide which stimulates adenylate cyclase: molecular cloning and characterization of the ovine and human cDNAs

A novel neuropeptide which remarkably stimulates adenylate cyclase in rat anterior pituitary cell cultures has been recently isolated from ovine hypothalami by A. Arimura and his collaborators (Biochem.Biophys.Res.Commun.164, 567-574(1989)). This peptide was designated as PACAP38(Pituitary Adenylate Cyclase Activating Polypeptide with 38 residues). In an attempt to investigate physiological implications of PACAP38, we have succeeded in cloning the cDNAs encoding the precursor of PACAP38 from ovine hypothalamus and human testis. An ovine cDNA encodes a protein of 176 amino acids in which PACAP38 is proceeded by a putative signal peptide and a "pro"-region (107 amino acids), and followed by a Gly-Arg-Arg sequence for proteolytic processing and amidation. Deduced amino-acid sequence of human PACAP38 was completely identical to that of the ovine isolated peptide. Cloning of PACAP38 cDNAs confirms the expression of the corresponding mRNAs and the presence of this neuropeptide in ovine hypothalamus and also in human testis.     

Cytoplasmic filaments in the endothelial cells of the sheathed capillary: an ultrastructural and immunocytochemical study in the pig spleen.

Cytoplasmic filaments of the endothelial cells of sheathed capillaries in the pig spleen were identified and their ultrastructure was studied. Two types of cytoplasmic filaments were found: intermediate filaments (diameter: 10 nm) which filled most of the interior of the cells, and thin filaments (diameter: 5 nm) which were located just beneath the cell membrane and filled the lateral cytoplasmic processes. In immunocytochemical preparations, the intermediate filaments were positive for vimentin and desmin, and were negative for keratin. Staining of the thin filaments with heavy meromyosin resulted in arrowhead formations. These observations suggest that the intermediate filaments maintain the cytoarchitecture, possibly protecting the cell from structural alterations induced by blood pressure changes. Concurrently, thin filaments may facilitate the passage of red blood cells and blood platelets through the interendothelial fenestrae of the sheathed endothelial cell to the reticular meshwork in the capillary sheath.     

Prothrombin Salakta: substitution of glutamic acid-466 by alanine reduces the fibrinogen clotting activity and the esterase activity.

Structural studies on a hereditary abnormal prothrombin, prothrombin Salakta, have been performed to identify the difference responsible for its reduced fibrinogen clotting activity and its reduced esterase activity. Amino acid composition and sequence analyses of a peptide isolated from a lysylendopeptidase digest of the abnormal thrombin indicated that Glu-466 had been replaced by Ala. This amino acid substitution can result from a single nucleotide change in the codon for Glu-466 (GAG----GCG). The model building and the molecular dynamics simulation of thrombin Salakta suggest that the Glu-466----Ala substitution would change the proper conformation around the substrate binding site containing Trp-468, which is a unique surface loop on the thrombin molecule. This is the experimental and theoretical evidence supporting the role of the surface loop containing Trp-468 for the proper conformation of the substrate binding site.