Differentiation of Langerhans Cells from Interdigitating Cells Using CD1a and S-100 Protein Antibodies

The present study shows that Langerhans cells can be differentiated from Interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.     

Matrix Gla protein C-terminal region binds to vitronectin. Co-localization suggests binding occurs during tissue development.

Matrix Gla protein (MGP) regulates calcification in cartilage and arteries. MGP synthesis during embryonic development and its binding and regulation of growth factors and morphogens of the TGF-beta/BMP superfamily suggests that it has additional functions. Assay by far-western gel overlays and gel filtration shift shows MGP binds vitronectin. Binding is saturable and consistent with a single class of binding sites. MGP binds to vitronectin but not collagen, fibromodulin, heparin, osteocalcin, chondroitin sulfate, laminin, ovalbumin or albumin. We have identified a vitronectin binding site within a 17-amino acid peptide 61-77 near the carboxyl-terminus that corresponds to a naturally occurring MGP C-terminus. MGP and the 61-77 MGP peptide also binds to fibronectin. MGP and vitronectin are focally co-localized in embryonic tissues. Co-localization in vivo suggests that the MGP and vitronectin interactions may modify cell-matrix interactions. Alternatively, vitronectin-bound MGP may have altered function for modulating BMP2 or TGF-beta activity. The current study demonstrates that MGP has a novel binding activity for vitronectin, an extracellular protein that promotes cell-matrix interactions and regulates coagulation.     

Breeding ofl-threonine hyper-producer ofEscherichia coli W

Summary l-Threonine hyper-producing mutants were obtained fromEscherichia coli W strain KY-8366, by reducingl-threonine degradation activity and enhancingl-threonine biosynthetic activity. Anl-threonine degradation reaction test using resting cells of KY-8366 suggested that the main pathway ofl-threonine degradation by KY-8366 is via glycine. A strain with reducedl-threonine degradation activity was obtained among those mutants that could not utilizel-threonine as sole nitrogen source. Rifampicin-resistant mutants andl-lysine plus methionine-insentitive mutants were isolated. These mutants showed enhanced aspartokinase levels and accumulated morel-threonine than the parental strains. Mutant H-4290 accumulated 58 g/l ofl-threonine.     

l-threonine production by l-aspartate- and l-homoserine-resistant mutant of Escherichia coli

Summary Growth and l-threonine productivity of l-threonine producer Escherichia coli H-4290 were inhibited by precursor amino acids, l-homoserine and l-aspartate. l-Threonine hyper-producers were isolated among the mutants resistant to l-homoserine and l-aspartate. Mutants H-4351 (Hom^r) and H-4578 (Hom^r, Asp^r) accumulated 22.2 g/l and 24.3 g/l of l-threonine in test tube cultures, while the parental strain H-4290 accumulated 18.2 g/l. The enzyme level of aspartokinase I (first enzyme of the threonine operon) was enhanced 2.3 times (H-4351) and 3 times (H-4578) that of H-4290. Mutant H-4578 accumulated 76 g/l of l-threonine in a 2-l jar fermentor after 75 h cultivation.Abbreviations DAP diaminopimeric acid - Met ^l poor growth in methionine-free medium - AHV -amino--hydroxyvaleric acid - Thr-N^- lack of ability to utilize l-threonine as a nitrogen source - Rif rifampicin - Lys+Met^r resistant to l-lysine and dl-methionine     

Roles of the respiratory Na+ pump in bioenergetics of Vibrio alginolyticus

Bioenergetic characteristics of Na+ pump-defective mutants of a marine bacterium Vibrio alginolyticus were compared with those of the wild type and revertant. Generation of membrane potential and motility at pH 8.5 in the mutants were completely inhibited by a proton conductor, carbonylcyanide m-chlorophenylhydrazone, whereas those in the wild type or revertant were resistant to the inhibitor. Motility and amino acid transport were driven by the electrochemical potential of Na+ not only in the wild type or revertant but also in the mutants. In the absence of the proton conductor, motility and amino acid transport of the mutants did not significantly differ from those of the wild type or revertant even at pH 8.5, where the Na+ pump has maximum activity. Therefore, the electrochemical potential of Na+ in the mutants seemed to be maintained at a normal level by a respiration-dependent H+ pump and Na+/H+ antiporter. On the other hand, growth of the mutants became defective as the medium pH increased, especially on minimal medium. These results indicate that the Na+ pump is an important energy-generating mechanism when nutrients are limited at alkaline pH.     

Evidence for unique homologous peptide sequences around the glycosylated seryl and threonyl residues in polysialoglycoproteins isolated from the unfertilized eggs of the Pacific salmon Oncorhynchus keta

Structures of glycopeptides obtained by exhaustive Pronase digestion of high molecular weight (1.7 X 10(5)) salmon egg polysialoglycoprotein have been elucidated. Six principal glycopeptides isolated by gel chromatography and DEAE-Sephadex A-25 chromatography in the absence or presence of borate ion were analyzed for their carbohydrate and amino acid composition, as well as amino acid sequence, and found to be of two distinct types: glycotripeptides, Thr*-Ser*-Glu, and glycotetrapeptides, Thr*-Gly-Pro-Ser, where an asterisk indicates the amino acid residues to which either the Gal beta 1----3GalNAc or Fuc alpha 1----3GalNAc beta 1----3Gal beta 1----4Gal beta 1----3GalNAc chain is attached. Their final yield corresponds to 64% of the original desialylated glycoprotein. In view of the simple amino acid composition of salmon egg polysialoglycoprotein (molar ratio Asp2Thr2Ser3Glu1Pro1Gly1Ala3) and the result of alkaline beta-elimination indicating three carbohydrate units linked to two of two threonine and one of three serine residues, a unique primary structure comprising repetitive sequences of the above two types of glycopeptides, which are interspersed by short nonglycosylated peptides consisting of alanine and aspartic acid, has been proposed for the core protein. The molecular secondary ion mass spectra of underivatized glycopeptides were used to obtain their structural information. The anomeric configuration of the proximal sugar-peptide linkages was proven to be alpha by proton nuclear magnetic resonance spectroscopy. This is the first systematic reported study of O-glycosidically linked glycopeptides by these instrumental methods.     

Purification and properties of extracellular glucosyltransferase synthesizing 1,6-, 1,3-alpha-D-glucan from Streptococcus mutans serotype a

An extracellular glucosyltransferase (sucrose: 1,6-, 1,3-alpha-D-glucan 3-alpha- and 6-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by DEAE-Sepharose chromatography and preparative isoelectric focusing. The molecular weight measured by SDS-PAGE was 159 000 and the isoelectric point was pH 4.9. The specific activity was 89.7 i.u. (mg protein)-1 and the optimum pH was 6.0. The Km value for sucrose was 4.9 mM and the enzyme activity was not stimulated by exogenous dextran T10. Glucan was synthesized de novo from sucrose by the purified enzyme and consisted of 49.1 mol% 1,6-alpha-linked glucose and 33.9 mol% 1,3-alpha-linked glucose, with 13.6 mol% terminal glucose and 3.3 mol% 1,3,6-alpha-branched glucose.     

Partial purification of two distinct enkephalin-degrading aminopeptidases from human cerebrospinal fluid

In human cerebrospinal fluid, aminopeptidase, dipeptidyl aminopeptidase, dipeptidyl carboxypeptidase, and carboxypeptidase which were capable of hydrolyzing enkephalins were detected. Among these enzymes, two distinct aminopeptidase, designated C-AP1 and C-AP2, were partially purified. These enzymes were not purified thoroughly, but the characteristics of C-AP2 were similar to those of an aminopeptidase purified from monkey brain. But the inhibitory activity of amastatin on C-AP2 was stronger, and that of substance P was negligible. On the other hand, characteristics of C-Ap1 were extremely differ from those of C-AP2 or an aminopeptidase purified from monkey brain. C-AP1 had an optimum pH more in the acidic range (the highest at pH 6.0) and was not inhibited by any of the protease inhibitor tested including bestatin and amastatin.     

Effects of dolichol and dolichyl phosphate on in vitro differentiation of hematopoietic progenitors

The exogenous addition of dolichyl phosphate (Dol-P), an active form of dolichol (Dol) that carries oligosaccharide chains for protein-N-glycosylation, significantly enhanced colony formation of mouse bone marrow hematopoietic progenitors (CFU-e, BFU-e, and CFU-gm) was stimulated by erythropoietin (Epo) and colony-stimulating factor (CSF), but Dol enhanced colony formation of CFU-e only. The effects of Dol or Dol-P on these hematopoietic progenitors were fully dependent on stimulation by Epo or CSF. Other mevalonate-metabolites, such as cholesterol, coenzyme Q10, and isopentenyladenine, had no effect on hematopoietic progenitors. These studies suggest that exogenous Dol-P enhances the frequency of differentiation of hematopoietic progenitors stimulated by Epo or CSF, and there may be a diversity in cellular response of these progenitors to Dol.