Synthesis of the mitogenic S-[2,3-bis(palmitoyloxy)propyl]-N-palmitoylpentapeptide from Escherichia coli lipoprotein

The N-terminal pentapeptide of the lipoprotein from the outer membrane of Escherichia coli was obtained by coupling S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteine to O-tert-butylseryl-O-tert-butyl-seryl-asparaginyl-alanine tert-butyl ester followed by deprotection with trifluoroacetic acid. The tetrapeptide was built up from alanine tert-butyl ester with N-9-fluorenylmethyloxycarbonyl protected amino acids. S-[2,3-Bis(palmitoyloxy)propyl]-N-palmitoylcysteine was obtained from N,N'-dipalmitoylcystine di-tert-butyl ester via reduction to the thiol, and S-alkylation with racemic 3-bromo-1,2-propanediol followed by esterification with palmitic acid in the presence of dicyclohexylcarbodiimide/dimethylaminopyridine and deprotection with trifluoroacetic acid. The compounds were characterized unequivocally by 13C-NMR and mass spectra. The diastereomers of S-[2,3-bis(palmitoyloxy)propyl]-N-palmitoylcysteine tert-butyl ester with opposite configuration at the propyl-C-2 atom could be separated on a silica-gel column.     

Melittin and a chemically modified trichotoxin form alamethicin-type multi-state pores

The bee venom constituent, melittin, is structurally and functionally related to alamethicin. By forming solvent-free planar bilayers of small area (approx. 100 microns 2) on the tip of fire-polished glass pipettes we could observe single melittin pores in these membranes. An increase in the applied voltage induced further non-integral conductance levels. This indicates that melittin forms multi-level pores similar to those formed by alamethicin. Trichotoxin A40, an antibiotic analogue of alamethicin, also induces a voltage-dependent bilayer conductivity, but no stable pore states are resolved. However, chemical modification of the C-terminal molecule part by introduction of a dansyl group leads to a steeper voltage-dependence and pore state stabilization. Comparing structure and activity of several natural and synthetic amphiphilic polypeptides, we conclude that a lipophilic, N-terminal alpha-helical part of adequate length (dipole moment) and a large enough hydrophilic, C-terminal region are sufficient prerequisites for voltage-dependent formation of multi-state pores.     

Influence of cholesterol content on red cell membrane viscoelasticity and fluidity.

The purpose of this investigation was to correlate the viscoelastic properties and lipid fluidity of the red blood cell membrane to its lipid composition. The viscoelastic properties of human red cells that had been enriched or depleted in cholesterol were determined by the micropipette technique. The lipid fluidity of the outer and inner leaflets of the erythrocyte membrane was concurrently assessed by steady state fluorescence depolarization. The elastic modulus and the viscosity moduli of the erythrocyte membrane showed no significant differences between the cholesterol-modified and the control cells. Cholesterol enrichment decreased the lipid fluidity of the outer membrane leaflet alone, and cholesterol depletion increased the fluidity mainly of the inner leaflet.     

Yeast mannans inhibit binding and phagocytosis of zymosan by mouse peritoneal macrophages

We have examined the effects of various mannans, glycoproteins, oligosaccharides, monosaccharides, and sugar phosphates on the binding and phagocytosis of yeast cell walls (zymosan) by mouse peritoneal macrophages. A phosphonomannan (PO(4):mannose ratio = 1:8:6) from kloeckera brevis was the most potent inhibitor tested; it inhibited binding and phagocytosis by 50 percent at concentrations of approximately 3-5 μg/ml and 10 μg/ml, respectively. Removal of the phosphate from this mannan by mild acid and alkaline phosphatase treatment did not appreciably reduce its capacity to inhibit zymosan phagocytosis. The mannan from saccharomyces cerevisiae mutant LB301 inhibits phagocytosis by 50 percent at 0.3 mg/ml, and a neutral exocellular glucomannan from pichia pinus inhibited phagocytosis by 50 percent at 1 mg/ml. Cell wall mannans from wild type S. cervisiae X2180, its mnn2 mutant which contains mannan with predominantly 1(arrow)6- linked mannose residues, yeast exocellular mannans and O-phosphonomannans were less efficient inhibitors requiring concentrations of 1-5 mg/ml to achieve 50 percent reduction in phagocytosis. Horseradish peroxidase, which contains high-mannose type oligosaccharides, was also inhibitory. Mannan is a specific inhibitor of zymosan binding and phagocytosis. The binding and ingestion of zymosan but not of IgG- or complement-coated erythrocytes can be obliterated by plating macrophages on substrates coated with poly-L-lysin (PLL)-mannan. Zymosan uptake was completely abolished by trypsin treatment of the macrophages and reduced by 50-60 percent in the presence of 10 mM EGTA. Pretreatment of the macrophages with chloroquine inhibited zymosan binding and ingestion. These results support the proposal that the macrophage mannose/N-acetylglucosamine receptor (P. Stahl, J.S. Rodman, M.J. Miller, and P.H. Schlesinger, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:1399-1403, mediates the phagocytosis of zymosan particles.     

A monoclonal antibody from infected mice to a Schistosoma mansoni egg proteinase

A number of monoclonal antibodies were obtained by fusion of SP2/0 myeloma cells and spleen lymphocytes from mice infected with Schistosoma mansoni. These antibodies were tested for their ability to inhibit acidic, thiol-dependent proteinases previously isolated from Schistosoma mansoni eggs and adult worms. One of the monoclonal antibodies isolated inhibits egg proteinase activity measured in vitro with the use of a low m.w. synthetic substrate. This antibody, which is an IgG1 isotype, does not appreciably inhibit an acidic, thiol-dependent proteinase obtained from the adult stage of Schistosoma mansoni. Immunocytochemical methods with the monoclonal antibody have been used to localize the egg proteinase within a set of "penetration" glands in the unhatched miracidium.     

Two high affinity estrogen binding proteins of different specificity in the immature rat uterus cytosol

The presence of two high affinity estrogen binding proteins in the uterine cytosol of the immature rat has been observed.Besides the 8 S cytosol estrogen $\text{receptor}$, there is a 4–5 S fraction binding estradiol and estrone with a large capacity. In fact, the two binding systems have a different affinity for estradiol and estrone, the receptor binding more the former and the 4–5 S fraction more the latter. Exposure of the cytosol to specific anti-α₁-Fetoprotein antibodies suppresses a large part of the 4–5 S binder, if not the totality. Moreover the estrogen binding 4–5 S fraction decreases with increasing age until puberty, while the $\text{receptor}$ increases. These results suggest therefore that the estradiol-estrone binding 4–5 S peak of the uterine cytosol is mainly made up of Estradiol Binding Plasma Protein-α₁-Fetoprotein (EBP-AFP). Also they confirm that “cytosol” should be taken as an operational fraction which may include extracellular components.During the course of these experiments, it has been observed that the increase of the estradiol $\text{receptor}$ is more rapid than the other uterine cytosol proteins until the 8th day, and that there is a second period of growth when it follows the development of the uterus and of the animal, as if it had reached a constant number of binding sites per cell.     

The Avena geo-curvature test

Summary The Avena geo-curvature test is a bioassay for auxin-type growth regulators. Etiolated oat coleoptiles are used and the test is conducted in special perspex trays under diffuse daylight. The coleoptiles, with the primary leaves intact, are arranged in grooves on the trays in 4 series of 6 coleoptiles each, and cut to a size of 10 mm. The test substance is applied on an agar strip covering only the lower halves of the apical cut surfaces of each series. After a 4-hour stay in the dark in moist Petri dishes the coleoptile cylinders are shadowgraphed on a 35-mm photographic film. The curvatures are measured from an enlarged projection (10 times natural size) of the shadowgraphs.The lowest IAA concentration which can be determined is around 30 to 60 g/l, i.e. about 1 to 2 ng IAA. The concentration response curves follow a logarithmic course up to 1,000 g/l. The standard error of the mean in a test series comprising 6 coleoptiles is on an average ± 7%. The simple and quick procedures make it possible for a worker to test 60 to 80 fractions a day.Dedicated to Professor Hans Söding on the occasion of his seventieth birthday. The senior author expresses his special gratitude for having been initiated by Professor Söding in the study of auxins.present address: Dpt. of Biology Princeton Univ. Princeton, N. J. 08540, U.S.A.     

Inactivation of bacteriophage, DNA, and ribonuclease by thermal hydrogen atoms

T1 phage, BU-T1 phage, infectious DNA extracted from phage phiX 174, and chromatographically purified ribonuclease were exposed to thermal hydrogen atoms, and the loss of plaque-forming ability, infectivity, or enzymatic activity was determined after various exposure times. Atomic hydrogen was generated by two different methods: (1) by a high-frequency discharge in hydrogen gas and (2) by irradiating a foil of polyethyleneter-ephthalate with 2-MeV protons. With increasing exposure time the surviving fraction of all objects tested approaches a constant level. After subtracting this constant "indestructible" fraction in either system, all objects were inactivated according to exponential curves. Furthermore, no BU sensitization was found to occur in BU-T1 phage exposed to atomic hydrogen, whereas gamma irradiation of samples from the same batches revealed a BU effect of a factor of 2.2. These experiments demonstrate hydrogen atoms to be efficient in causing biological damage. Consequently the terminology of "direct" and "indirect" radiation effect may have to be redefined.