Aerial oxidation of a redox-active porphyrin in the presence of a spin trap

Aerial oxidation of meso-tetrakis(3,5-dimethoxy-4-hydroxyphenyl)- porphyrin 3, in the presence of the water-soluble spin trap (4-pyridyl-1-oxide)-N-t-butylnitrone (POBN), gives the porphyrin radical 4, in which spin density is localized on a phenoxyl meso-substituent. Evidence is presented to show that the spin trap inhibits solution aggregation and spin exchange of 4, but does not, as originally expected, form spin adducts with reduced-oxygen species.     

Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units.     

The human placental protein 14 (PP14) gene is localized on chromosome 9q34

Summary PP14 protein (placental protein 14) is abundantly secreted by the human endometrium under the influence of progesterone. Human PP14 is homologous to -lactoglobulin, the main component of equine, bovine, and ovine milk whey. A genomic PP14 probe (PP14G1) was used for the chromosome assignment of the PP14 gene. Somatic hybrid cells enabled PP14G1 to be assigned to chromosome 9. In situ hybridization further refined this assignment to 9q34. The localization of the PP14 gene in the region of the ABO locus is consistent with the linkage described in bovines between beta-lactoglobulin and the J blood group (homologous to the human ABO group). Offprint requests to: V.C. Nguyen     

A change in twist of actin provides the force for the extension of the acrosomal process in limulus sperm: the false-discharge reaction

One of the most spectacular motions is the generation of the acrosomal process in the limulus sperm. On contact with the egg, the sperm generates a 60-mum-long process that literally drills its way through the jelly surrounding the egg. This irresversible reaction takes only a few seconds. We suggested earlier that this motion is driven by a change in twist of the actin filaments comprising the acrosomal process. In this paper we analyze the so-called false discharge, a reversible reaction, in which the acrosomal filament bundle extends laterally from the base of the sperm and not anteriorly from the apex. Unlike the true discharge, which is straight, the false discharge is helical. Before extension, the filament bundle is coiled about the base of the sperm. In the coil, the bundle is not smoothly bent but consists of arms (straight segments) and elbows (corners) so that the coil looks like a 14-sided polygon. The extension of the false discharge works as follows: starting at the base of the bundle, the filaments change their twist which concomitantly changes the orientations of the elbows relative to each other; that is, in the coil, the elbows all like in a common plane, but after the change in twist, the plane of each elbow is rotated to be perpendicular to that of its neighbors. This change transforms the bundle from a compact coil into an extended left- handed helix. Because the basal end of the bundle is unconstrained, the extension is lateral. The true discharge works the same way but starts at the apical end of the bundle. The apical end, however, is constrained by its passage through the nuclear canal, which directs the extention anteriorly. Unlike the false discharge, during the true discharge the elbows are melted out, making the reaction irreversible. This study shows that rapid movement can be regenerated by actin without myosin and gives us insight into the molecular mechanism.     

The cytoskeleton and the cellular traffic of the progesterone receptor

Previous studies on glucocorticoid receptors have suggested the existence of interactions between the receptor and microtubule or actin networks. It was hypothesized that such interactions may contribute to the guidance of steroid hormone receptors towards the nucleus. We used a permanent L cell line expressing the delta 638-642 progesterone receptor. This mutant has all the characteristics of the wild type receptor except that the deletion of five amino acids inactivates the constitutive karyophilic signal. Consequently, the receptor is cytoplasmic in the absence of hormone but is shifted into the nucleus when administration of hormone activates the second karyophilic signal. Optical microscopy and confocal laser microscopy were used in intact cells or in cells depleted of soluble elements by permeabilization with detergents. By immunofluorescence, the receptor was found to be mainly concentrated in the perinuclear area. A small fraction of progesterone receptor (PR) persisted in this region after Triton X100 treatment. These observations suggested that the receptor could interact with some insoluble constituent(s) of the cytoplasm. However, careful colocalization studies showed that this heterogenous distribution was not due to interactions with microtubule, microfilament, or intermediate filament networks. Functional involvement of these networks in the translocation of the receptor into the nucleus was studied after cell treatment with cytoskeletal drugs such as nocodazole, demecolcine and cytochalasin. None of these compounds prevented or even delayed the hormone-dependent transfer of delta 638-642 PR into the nucleus. Similar conclusions were reached with the wild type receptor expressed by transfection in Cos-7 cells. PR was shifted from the nucleus into the cytoplasm by administration of energy-depleting drugs. After disruption of the various cytoskeletal networks normal nuclear reaccumulation of the receptor was observed when these drugs were removed. The results thus suggest that the progesterone receptor is not colocalized with the main cytoskeletal components. Disruption of the cytoskeletal networks does not prevent its nuclear translocation. Thus, karyophilic signals and interactions with the nuclear pore seem to be the primary determinants of the cellular traffic of the progesterone receptor.     

Mechanism and kinetics of the thermal activation of glucocorticoid hormone receptor complex.

Steroid-receptor complexes formed at low temperature and ionic strength do not bind to nuclei or chromatin. After a temporary exposure to high temperature, or ionic strength, or both, a fraction of them becomes activated (able to bind to nuclei). An assay of the activated form of the complex based upon titration with nuclei in excess was established. This assay was used to perform kinetic and equilibrium studies of the thermal activation of glucocorticoid-receptor complex in order to elucidate its mechanism. It was found that the reaction is of apparent first order and yields a monomolecular product. It thus probably consists of a conformational change in the steroid-receptor complex. The rate of activation is 1.37 +/- 0.06 X 10(-3) S-1 at 25 degrees. The free energy of thermodynamic activation (The word activation is used here in its usual thermodynamic meaning and not in the sense of receptor modification) of this reaction is greater than G = 21.3 Kcal. The corresponding enthalpy and entropy are respectively greater than H = 31.4 kcal and greater than S = 4 cal/degree. These positive and high values of greater than H and greater than S are very similar to those described for denaturation reactions of proteins suggesting that breakage of some noncovalent bonds could take place during activation. The reaction proceeds until approximately 60% of the complexes are activated. It was shown that this corresponds to an equilibrium between activated and nonactivated forms and not to the presence of a population of complexes unable to undergo activation. This equilibrium is not modified by temperature variations between 10 degrees and 30 degrees. It is possible to activate over 80% of the complexes when the activation is performed in the presence of excess acceptor, thus shifting the equilibrium. A similar situation is probably observed in situ in cells since 90% of the complexes are found in the nuclei when liver slices are incubated with hormone.