Three states for the formyl peptide receptor on intact cells

Three distinct states of the formyl peptide receptor have been described. These are: 1) the ternary complex of ligand, receptor, and G protein (LRG); 2) the rapidly dissociating occupied receptor (ligand-receptor complex (LR]; and 3) a desensitized slowly dissociating guanine nucleotide-insensitive receptor (desensitized ligand-receptor complex ("LRX"]. During cell activation there is a rapid interconversion among receptor states from a rapidly dissociating form (t 1/2 approximately 10 s) to a slowly dissociating form (t 1/2 greater than or equal to 2 min). Neither the dynamics of the states nor their interconversion is influenced by ribosylation of G protein in the presence of pertussis toxin. In contrast to ribosylation, treatment of cells with either 2-deoxyglucose or fluoride ion, both of which lead to a loss of adenine and guanine nucleotides, causes a time-dependent change in ligand dissociability. After short periods of treatment (5-15 min) rapid dissociation is observed; after longer times (30-60 min), slow dissociation is once again detected. When intact cells are first ribosylated and then energy-depleted, only a rapidly dissociating receptor is detected. These results are discussed in terms of a model with the following elements: 1) intact cell dynamics during cell activation are dominated by an energy-dependent interconversion from LR to LRX; 2) under activation conditions, LRG appears and disappears too rapidly to be detected; 3) in cells depleted of energy and guanine nucleotide, LRG is stabilized; 4) in cells both ribosylated and depleted of energy, LR is stabilized.     

Palmitylation of the glycoprotein IIb-IIIa complex in human blood platelets

The presence of covalently bound palmitic acid in fibrinogen receptors, glycoproteins (GP) IIb and IIIa, has been explored in human blood platelets. Membrane fractions were isolated from fresh blood platelets labeled with [9,10-3H]palmitic acid and then analyzed for radioactive proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands were visualized by staining with Coomassie Brilliant Blue, excised, and counted in a liquid scintillation counter. The results indicate that membrane proteins with electrophoretic mobility corresponding to glycoproteins IIb and IIIa incorporate [9,10-3H]palmitic acid. The palmitylated glycoproteins IIb and IIIa were immunoprecipitated by specific anti-GP IIb and GP IIIa antisera. It is interesting to note that the palmitylation of these glycoproteins occurred rapidly in platelets activated with 0.5 unit of thrombin or 30 microM ADP. At the concentration used (100 micrograms/ml), cycloheximide did not inhibit incorporation of [3H]palmitate into the glycoproteins showing that this process is not dependent upon protein synthesis. The acyl moiety was resistant to denaturating detergents, delipidation with organic solvents, and hydrolyzable with hydroxylamine. In the case of membrane protein with the electrophoretic mobility of GP IIb, the radioactive label was significantly decreased after reduction with 2-mercaptoethanol. Final identification of GP IIIa as an acylated product in human platelets incubated with [9,10-3H]palmitic acid was provided by two-dimensional polyacrylamide gel electrophoresis. In contrast to GP IIb alpha, GP IIIa isolated by this method showed the presence of attached radioactive palmitic acid residues. Analysis by high performance liquid chromatography after methanolysis of the [3H]palmitate-labeled glycoproteins confirmed the fatty acid nature of the label. Palmitylation is a newly identified post-translational modification of the fibrinogen receptor which may play an important role in its interaction with the membrane and/or its biological function.     

Dehydrodolichyl diphosphate synthase in Sertoli and spermatogenic cells of prepuberal rats

The specific activity of 2,3-dehydrodolichyl diphosphate synthase in homogenates of protease-treated seminiferous tubules, enriched spermatogenic cells, and Sertoli cells changed as a function of the age of prepuberal rats. The highest enzymatic activity occurred in each case in 23-day-old rats. Homogenates of pachytene spermatocytes, spermatids, or Sertoli cells had higher synthase activity than a whole testicular homogenate prepared by protease treatment of tubules. Enzymatic activity in pachytene spermatocytes expressed per mg of protein was about 1.7-fold higher than in spermatids, 5.3-fold higher than in spermatogonia, and about 8.3-fold higher than in spermatozoa. Therefore, the increase in spermatogenic cell synthase before day 23 can be accounted for by the appearance of the pachytene spermatocytes. Enzymatic activity decreased remarkably after the differentiation of spermatids into spermatozoa. Synthase activity in enriched Sertoli cell preparations was 1.5-2.3-fold higher than in spermatogenic cell preparations between days 15 and 30. Therefore, both spermatogenic cells and Sertoli cells contribute to changes in the enzymatic activity in seminiferous tubules during development. These changes may be important in regulating the availability of dolichyl phosphate for glycoprotein synthesis during early stages of differentiation.     

Acetylcholine receptor in a C2 muscle cell variant is retained in the endoplasmic reticulum

We have examined the properties and intracellular localization of acetylcholine receptors in the C2 muscle cell line and in a variant (T-) that accumulates AChR intracellularly. On immunoblots, the subunit structures of the AChR from wild-type and T- cells were similar except that the gamma and delta subunits of the variant AChR had altered mobilities. Digestion with endoglycosidases H and F demonstrated that this difference results from a failure of high-mannose N-linked oligosaccharides on AChR subunits to be processed to complex forms in the variant. N-linked glycosylation of other proteins in the variant was normal. When examined by immunocytochemistry, the distribution of internal AChR in wild-type cells was consistent with a location both in the endoplasmic reticulum and in the Golgi. Variant cells, however, showed no evidence of Golgi staining. Subcellular fractionation experiments also demonstrated AChR in the Golgi fractions of wild-type cells, but not in those derived from T- cells. We conclude that in T- myotubes most of the AChR fails to be transported out of the endoplasmic reticulum.     

Polymorphic repetitive DNA sequences in Mycobacterium tuberculosis detected with a gene probe from a Mycobacterium fortuitum plasmid

Clinical isolates of Mycobacterium tuberculosis were shown by Southern blotting to contain DNA sequences hybridizing to a probe derived from a Mycobacterium fortuitum plasmid. Two such M. tuberculosis DNA fragments, isolated from a gene library, were used as probes to show restriction fragment length polymorphism in M. tuberculosis strains by detecting a repetitive sequence apparently located at different points on the chromosome. This could indicate the presence of a transposable element in M. tuberculosis which is partly homologous to a region of the M. fortuitum plasmid. The probes described can be used to fingerprint M. tuberculosis isolates, and in addition are capable of distinguishing M. tuberculosis from Mycobacterium bovis and BCG.     

Production of a retroviral P15E-related chemotaxis inhibitor by IL-1-treated endothelial cells. A possible negative feedback in the regulation of the vascular response to monokines

The effects of IL-1 on vascular endothelium result in a complex set of alterations which are potentially disruptive of vessel wall and underlying tissue integrity. The present study was aimed at investigating possible regulation of such potentially destructive responses elicited by IL-1 on endothelial cells. Culture supernatants of IL-1-treated human umbilical vein endothelial cells (HEC) were depleted of retroviral p15E-related Ag with immobilized anti-p15E mAb. The monocyte chemotactic and polarizing activity of supernatants of IL-1-treated HEC (presumably related to colony-stimulating factors being released by HEC) was markedly augmented by absorption on immobilized anti-p15E antibodies. Irrelevant IgG had no effect and anti-p15E antibodies did not affect the chemotactic activity of supernatants from unstimulated HEC. The material eluted from Sepharose-bound anti-p15E antibodies was devoid of chemotactic and polarizing activity and suppressed the polarization and migration of monocytes in response to chemoattractants. The alpha and beta molecular species of IL-1 were equally effective in inducing the production of p15E-related inhibitor. The production of a p15E-related inhibitor of chemotaxis induced by IL-1 in HEC may represent a negative signal in the regulation of the potentially destructive responses to pro-inflammatory cytokines.     

Fatty acid composition of lipopolysaccharides of the strains of different species of Yersinia

The fatty acid composition of lipopolysaccharides of the strains of Y. enterocolitica, Y. intermedia, Y. frederiksenii and Y. ruckeri studied during cultivation on meat-peptone agar is characterized by the predominance of 3-hydroxytetradecanoic and dodecanoic acids. Closely related to the mentioned bacteria is the strain of Y. kristensenii which is distinguished only by its higher level of hexadecanoic acid. The strains of Y. pseudotuberculosis and the vaccine strain of Y. pestis have a uniform fatty acid composition of lipopolysaccharides with predominance of 3-hydroxytetradecanoic acid. Their relatively low level of dodecanoic acid conditions the characteristic fatty acid spectrum of lipopolysaccharides which differs from that of the above mentioned group of Yersinia. The peculiarities of the fatty acid composition of lipopolysaccharides of both groups of Yersinia are preserved during growth on meat-peptone broth, but the increase in the level of hexadecanoic acid balances the differences between Y. kristensenii, the other Y. enterocolitica-like bacteria and Y. ruckeri. The obtained results confirm close relationship of Y. pseudotuberculosis and Y. pestis, and also of Y. enterocolitica and Y. enterocolitica-like bacteria, showing propinquity of Y. ruckeri to the latter.     

A micromorphological study of some representative genera in the tribe Saturejeae (Lamia ceae)

HUSAIN, S. Z., MARIN, P. D., ŠILIĆ, Č., QAISER, M. & PETCOVIĆ, B., 1990. A micromorphological study of some representative genera in the tribe Saturejeae (Lamiaceae). The Old World genera in the tribe Saturejeae are usually distributed either in Europe and North Africa or in the temperate parts of Asia. The centres of distribution of investigated genera are mainly in the Mediterranean region. In taxonomic revisions very little reference is made to micromorphological characters, in particular, to nutlets and leaf indumentum, in spite of the stability of these characters. Scanning electron microscopy of nutlet surface and patterns of leaf indumentum show a wide range of variation, not only among genera, but also at lower levels of classification. In view of this, nutlet surface and leaf indumentum structure, as seen with the SEM, of representative species of eight genera in the tribe Saturejeae provides useful additional character combinations in delimiting these closely related genera. This study also supports Boissier's delimitation of sections Micromeria and Pseudomelissa.     

Use of rat primary lung cells for studying genotoxicity with the sister-chromatid exchange and micronucleus assays

Primary culture of lung cells from CD rats was established for pulmonary genotoxicity studies using two genetic endpoints, sister-chromatid exchange (SCE) and micronucleus formation (MN). In the cell isolation study, a combined enzyme separation of rat lungs with trypsin (1.3 mg/ml) plus collagenase (50 U/ml) gave the highest yield of viable and colony-forming cells. For the MN assay, the cytokinesis block induced by cytochalasin B (CYB) was employed to enumerate MN in binucleated (BN) cells. Treatment of primary lung cells with 2 micrograms CYB/ml for two days appeared to be optimal for scoring micronuclei in CYB-induced BN cells. By this procedure, mitomycin C (MMC), triethylenemelamine, and benzo[a]pyrene caused a dose-related increase in micronucleated BN cells in vitro without metabolic activation. In the SCE assay, maximum second-division metaphases were obtained after cells were incubated with bromodeoxyuridine for 48-54 h. After this incubation time, high frequencies of SCE induced by MMC and 3-methylcholanthrene after in vitro exposure (without S9 activation) or in vivo exposure were observed. The results indicate that rat primary lung cells can metabolize polycyclic aromatic hydrocarbons and that this lung cell system is potentially useful for the detection of pulmonary genotoxicants.     

Net Mg2+ uptake in relation to the amount of exchangeable Mg2+ in the Donnan free space of ryegrass roots

Ammonium acetate and BaCl₂-triethanolamine were used to desorb Mg²⁺ from the root Donnan free space (DFS) of 23-d-old ryegrass (Lolium multiflorum Lam. cvs. Gulf and Wilo). Amounts of desorbed Mg²⁺ increased with the increase in Mg²⁺ activity of the nutrient solution. Slightly less Mg²⁺ was desorbed by Ba²⁺ than by NH₄ ⁺. Previously published data on short-term net Mg²⁺ uptake by intact 23-d-old ryegrass plants of the two cultivars were linearly related to the amount of exchangeable Mg⁺ desorbed from the root DFS (r²=0.90 and 0.81 for the desorption by NH₄ ⁺ and Ba²⁺, respectively). A sward of Mg²⁺ ions attracted to the negative charges of the cell surface is suggested to represent a part of a pool of Mg²⁺ available for active transport through the plasmalemma.