Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.     

The extracellular domain of p185/neu is released from the surface of human breast carcinoma cells, SK-BR-3.

The human breast carcinoma cell line SK-BR-3, expresses the neu oncogene product, p185, which is a receptor tyrosine kinase. Using a double monoclonal antibody capture enzyme-linked immunosorbent assay for p185, activity was detected in conditioned media from cultures of SK-BR-3 cells. Two monoclonal antibodies specific for the extracellular domain of p185/neu immunoprecipitated a protein with a molecular mass of approximately 105 kDa. p105 was further shown to compete with p185 for binding to monoclonal antibodies and pulse-chase experiments indicate that it was generated by post-translational processing. Peptide maps showed that p105 and p185 are related polypeptides. Since p105 is close to the predicted size for the extracellular domain of p185/neu, we propose that SK-BR-3 cells specifically process and release this portion of the receptor into the medium. The release of the extracellular domain may have implications in oncogenesis and its detection could prove useful as a cancer diagnostic.     

Genes for tryptophan biosynthesis in the halophilic archaebacterium Haloferax volcanii: the trpDFEG cluster.

Tryptophan auxotrophs of the archaebacterium Haloferax volcanii define a cluster of overlapping genes homologous to eubacterial-eukaryotic trpD, -F, -E, and -G, linked in that order and each preceded by a possible ribosome binding site. Residues involved in feedback inhibition of eubacterial anthranilate synthetases are conserved.     

Elucidation of the complete Azorhizobium nicotinate catabolism pathway.

A complete pathway for Azorhizobium caulinodans nicotinate catabolism has been determined from mutant phenotype analyses, isolation of metabolic intermediates, and structural studies. Nicotinate serves as a respiratory electron donor to O2 via a membrane-bound hydroxylase and a specific c-type cytochrome oxidase. The resulting oxidized product, 6-hydroxynicotinate, is next reduced to 1,4,5,6-tetrahydro-6-oxonicotinate. Hydrolytic ring breakage follows, with release of pyridine N as ammonium. Decarboxylation then releases the nicotinate C-7 carboxyl group as CO2, and the remaining C skeleton is then oxidized to yield glutarate. Transthioesterification with succinyl coenzyme A (succinyl-CoA) yields glutaryl-CoA, which is then oxidatively decarboxylated to yield crotonyl-CoA. As with general acyl beta oxidation, L-beta-hydroxybutyryl-CoA, acetoacetyl-CoA, and finally two molecules of acetyl-CoA are produced. In sum, nicotinate is catabolized to yield two CO2 molecules, two acetyl-CoA molecules, and ammonium. Nicotinate catabolism stimulates Azorhizobium N2 fixation rates in culture. Nicotinate catabolism mutants still able to liberate pyridine N as ammonium retain this capability, whereas mutants so blocked do not. From, mutant analyses and additional physiological tests, N2 fixation stimulation is indirect. In N-limited culture, nicotinate catabolism augments anabolic N pools and, as a consequence, yields N2-fixing cells with higher dinitrogenase content.     

Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution.

The majority of Pseudomonas aeruginosa strains synthesize two antigenically distinct types of lipopolysaccharide (LPS), namely, a serotype-specific B-band LPS and a common antigen A-band LPS. A-band LPS consists of uncharged poly-D-rhamnan, which does not bind uranyl ions and is difficult to stain for electron microscopy; the highly charged B-band LPS is more easily visualized. We selected two wild-type strains, PAO1 (serotype O5) and IATS O6 (serotype O6), generated isogenic mutants from them, and examined the distribution of LPS on the surface of these organisms by freeze-substitution and electron microscopy. On PAO1 cells, which express both A-band and B-band LPSs, a 31- to 36-nm-wide fringe extending perpendicularly from the outer membrane was observed. A fine fibrous material was also observed on the surface of serotype O6 (A+ B+) cells, although this material did not form a uniform layer. When the LPS-deficient mutants, strains AK1401 (A+ B-), AK 1012 (A- B-), rd7513 (A- B-), and R5 (an IATS O6-derived rough mutant; A- B-), were examined, no extraneous material was apparent above the bilayer. However, an asymmetrical staining pattern was observed on the outer leaflet of the outer membrane of each of these mutants, presumably conforming to the anionic charge distribution of the core region of the rough LPS. In all cases, expression of the LPS types was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. When optical densitometry on electron microscopy negatives was used to analyze the outer membrane staining profiles, subtle differences in the degrees of core deficiency among rough mutants were detectable. This is the first time an electron microscopy technique has preserved the infrastructure produced in the outer membrane by its constituent macromolecules. We conclude that freeze-substitution electron microscopy is effective in the visualization of LPS morphotypes.     

Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells.

Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin. Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0-72 hr with or without LPS and then measuring ACE activity. ACE activity in intact HUVEC monolayers incubated with LPS (10 micrograms/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells. The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS. The LPS-induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml. That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane. While some LPS serotypes (Escherichia coli 0111:B4 and 055:B5, S. minnesota) were more potent inhibitors of ACE activity than others (E. coli 026:B6 and S. marcescens), all LPS serotypes tested were inhibitory. These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.     

Isolation and characterization of a chymotryptic fragment of platelet glycoprotein IIb-IIIa retaining Arg-Gly-Asp binding activity.

Platelet membrane glycoprotein (GP)IIb-IIIa exists as a divalent cation-dependent heterodimer which recognizes the Arg-Gly-Asp (RGD) sequence of adhesive proteins. To isolate the RGD binding domain of GPIIb-IIIa we performed proteolysis of GPIIb-IIIa with alpha-chymotrypsin. GPIIb-IIIa was bound to an affinity matrix of GRGDSPK-coupled Sepharose 4B and was then treated with chymotrypsin. After washing the unbound fragments, two discrete polypeptides of 55 and 85 kDa remained bound to the RGD affinity matrix and were specifically eluted by soluble HHLGGAKQAGDV (H12) or by GRGDSP, but not by GRGESP. Immunoblotting with subunit-specific polyclonal antibodies showed that the 55- and 85-kDa fragments were derived from GPIIb and GPIIIa, respectively. Amino-terminal sequencing and immunoblotting using site-specific antibodies indicated that these fragments contained the amino termini of their parent molecules. In the presence of 1 mM Ca2+ and 1 mM Mg2+, these two fragments were maintained as a heterodimer inasmuch as both fragments were immunoprecipitated by the polyclonal anti-GPIIIa antibodies. In contrast, chelating the divalent cations with 5 mM EDTA resulted in the lack of co-immunoprecipitation of the 55-kDa GPIIb fragment. After removal of the H12 peptide, the 55/85-kDa heterodimer bound to immobilized fibrinogen in an enzyme-linked immunosorbent assay by an RGD-dependent mechanism. These findings suggest that the RGD binding domain and structures required for heterodimer maintenance are present within the 55/85-kDa chymotryptic fragment of GPIIb-IIIa.     

Evidence of two isomers of phosphatidylinositol in plant tissue

The structure of phosphatidylinositol in barley (Hordeum vulgare) aleurone layers was investigated by chemical degradation. In vivo myo-[2-³H]inositol-labeled phosphatidylinositol was first converted to glycerophosphoinositol and, subsequently, after removal of the glycerol moiety, to inositol monophosphate. Here, we present data that show that, in addition to the commonly occurring 1,2-diacylglycero-3-(d-myo-inositol-1-phosphate), barley aleurone cells contain a novel second isomer of phosphatidylinositol that differs in structure of the head group.     

Presence of immunoreactive endothelin in human milk

Endothelin-like immunoreactivity was detected in human milk at a concentration of 6.8 +/- 1.6 pmol/l (mean +/- SEM; n = 16) using a highly sensitive radioimmunoassay. Gel filtration and fast protein liquid chromatography (FPLC) verified the identity of the endothelin. FPLC revealed 4 peaks, one eluting just after the void volume, and the other three in the positions of endothelin-1, -2, and -3, respectively.