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.     

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.     

Typing of Nontypable Staphylococci by Lysogeny

Strains of coagulase-positive staphylococci which were nontypable with the routine typing set of phages could be typed by lysogeny with phage-propagating strains as indicators and with ultraviolet induction. About 10% of the strains could be typed without induction. About 36% of them could be typed by this method when ultraviolet irradiation was used as an inducing agent. The phage groups from which the majority of the nontypable staphylococci originated were easily identified by this method of typing.     

Rapid modulation of homing receptors (gp90MEL-14) induced by activators of protein kinase C. Receptor shedding due to accelerated proteolytic cleavage at the cell surface

Lymphocyte entry into lymph nodes and Peyer's patches is initiated by the adhesion of the lymphocytes to specialized postcapillary high endothelial venules (HEV). The binding of lymphocytes to lymph node HEV is mediated by the cell surface receptor gp90MEL-14 (gp90). Previous work has shown that gp90 is down-regulated over a period of days after mitogenic or mixed lymphocyte reaction stimulation of T lymphocytes. In our study, it is shown that stimulation of lymphocytes with activators of protein kinase C (PKC), such as PMA or 1-oleoyl 2-acetyl-glycerol, results in the nearly complete loss of surface expression of gp90 within 1 h. Pretreatment of the cells with H-7 or staurosporine, PKC inhibitors, but not HA1004, a general protein kinase inhibitor, prevents the loss of gp90MEL-14. Within 15 min of stimulation of PKC, a novel form of gp90 can be immunoprecipitated from the supernatant of stimulated cells. Upon deglycosylation, this soluble gp90 polypeptide is shown to be 12 kDa smaller than the cell surface protein. Peptide mapping showed identical patterns for surface and soluble receptor, confirming that the soluble Ag is related to the cell membrane protein. Together, these experiments suggest that activation of PKC results in the proteolytic cleavage of gp90MEL-14, resulting in receptor shedding and the inability of the lymphocytes to adhere to HEV endothelium. Furthermore, because supernatant from unstimulated, normal lymphocytes also contains a small amount of the low Mr form of gp90, cell surface proteolysis may be part of the normal turnover of this receptor glycoprotein. These experiments suggest that PKC may play a role in the regulation of lymphocyte traffic to lymphoid tissues.