The ferrous-oxy complex of human aromatase

In this communication, we document the self-assembly of heterologously expressed truncated human aromatase (CYP19) into nanometer scale phospholipids bilayers (Nanodiscs). The resulting P450 CYP19 preparation is stable and can tightly associate with the substrate androstenedione to form a nearly complete high-spin ferric protein. Ferrous CYP19 in Nanodiscs was mixed anaerobically in a rapid-scan stopped-flow with atmospheric dioxygen and the formation of the ferrous-oxy complex observed. First order decay of the oxy-complex to release superoxide and regenerate the ferric enzyme was monitored kinetically. Surprisingly, the ferrous-oxy complex of aromatase is more stable than that of hepatic CYP3A4, opening the path to precisely determine the biochemical and biophysical properties of the reaction cycle intermediates in this important human drug target.     

PI 3-kinases and Src kinases regulate spreading and migration of cultured VSMCs

Pulmonary artery smooth muscle cell (PASMC)adhesion, spreading, and migration depend on matrix-stimulatedreorganization of focal adhesions. Platelet-derived growth factor(PDGF) activates intracellular signal transduction cascades that alsoregulate adhesion, spreading, and migration, but the signalingmolecules involved in these events are poorly defined. We hypothesizedthat phosphatidylinositol (PI) 3-kinases and Src tyrosine kinasestranslate matrix and PDGF-initiated signals into cell motility. Inexperiments with cultured canine PASMCs, inhibition of PI 3-kinaseswith wortmannin (0.3 µM) and LY-294002 (50 µM) and of Src kinasewith PP1 (30 µM) did not decrease spontaneous (nonstimulated) orPDGF-stimulated (10 ng/ml) adhesion onto collagen. PI 3-kinase and Srckinase activities, however, were necessary for cell spreading: PP1inhibited cell spreading and Src Tyr-418 phosphorylation in aconcentration-dependent manner. Inhibition of PI 3-kinase and Srcpartially reduced cell migration, while at 10 and 30 µM, PP1eliminated migration, likely due to inhibition of PDGF receptors. Inconclusion, both PI 3-kinases and Src tyrosine kinases are componentsof pathways that mediate spreading and migration of cultured PASMCs on collagen.

     

Frog lysozyme III. The effects of metamorphic inhibition on Rana pipiens lysozyme ontogeny.

The inhibition of Rana pipiens metamorphosis by thiouracil altered the ontogeny of lysozyme. Certain isozymes of the enzyme remained absent. There was, nevertheless, an increase in tissue lysozyme concentration.     

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t_1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.     

Infrared spectra of outer and cytoplasmic membranes of Escherichia coli

Outer and cytoplasmic membranes of Escherichia coli were prepared by a method based on isopyenic centrifugation on a sucrose gradient. The infrared spectra of solid films of these membranes were studied. The cytoplasmic membrane had an amide I band at 1657 cm^?1 and an amide II band at 1548 cm^?1. The outer membrane had a broad amide I band at 1631–1657 cm^?1 and an amid II band at 1548 cm^?1 with a shoulder at 1520–1530 cm^?1. Upon deuteration, the amide I band of the cytoplasmic membrane shifted to 1648 cm^?1, whereas the band at 1631 cm^?1 of the outer membrane remained unchanged. After extraction of lipids with chloroform and methanol, the infrared spectra in the amide I and amide II regions of both membranes remained unchanged. Although the outer membrane specifically contained lipopolysaccharide, this could not account for the difference in the infrared spectra of outer and cytoplasmic membranes. It is concluded that a large portion of proteins in the outer membrane is a β-structured polypeptide, while this conformation is found less, if at all in the cytoplasmic membrane.     

Effect of α-crystallin on thermal aggregation of glycogen phosphorylase <Emphasis Type="Italic">b</Emphasis> from rabbit skeletal muscle

Thermal aggregation of rabbit skeletal muscle glycogen phosphorylase b (Phb) has been investigated using dynamic light scattering under conditions of a constant rate of temperature increase (1 K/min). The linear behavior of the dependence of the hydrodynamic radius on temperature for Phb aggregation is consistent with the idea that thermal aggregation of proteins proceeds in the kinetic regime wherein the rate of aggregation is limited by diffusion of the interacting particles (the regime of "diffusion-limited cluster-cluster aggregation"). In the presence of alpha-crystallin, a protein exhibiting chaperone-like activity, the dependence of the hydrodynamic radius on temperature follows the exponential law; this suggests that the aggregation process proceeds in the kinetic regime where the sticking probability for colliding particles becomes lower than unity (the regime of "reaction-limited cluster-cluster aggregation"). Based on analysis of the ratio between the light scattering intensity and the hydrodynamic radius of Phb aggregates, it has been concluded that the addition of alpha-crystallin results in formation of smaller size starting aggregates. The data on differential scanning calorimetry indicate that alpha-crystallin interacts with the intermediates of the unfolding process of the Phb molecule. The proposed scheme of thermal denaturation and aggregation of Phb includes the stage of reversible dissociation of dimers of Phb into monomers, the stage of the formation of the starting aggregates from the denatured monomers of Phb, and the stage of the sticking of the starting aggregates and higher order aggregates. Dissociation of Phb dimer into monomers at elevated temperatures has been confirmed by analytical ultracentrifugation.     

Comparative study of spectral and antioxidant properties of pigments from the eyes of two Mysis relicta (Crustacea, Mysidacea) populations, with different light damage resistance

Retinal visual and screening pigments of two populations (one marine and the other freshwater) of the opossum shrimp Mysis relicta Lovén (Crustacea, Mysidacea), which have different ocular tolerance to light, was investigated. Visual pigments were extracted by detergent and their bleaching difference spectra were determined. The difference between the visual pigment absorption maximum of the two populations correlated with their difference in spectral sensitivity. Using buffer or neutral methanol, a yellow pigment was extracted which had absorption maxima at 440 nm and 325 nm and bright blue fluorescence (λ_max 415 nm). A screening pigment (ommochrome) with maximum at 525 nm was extracted by acid methanol, and was probably related to the group of ommines. The eyes of the lake population had 1.8–2.7 times less of this pigment than the eyes of the sea population. The sea population is more resistant to photo-induced accumulation of thiobarbituric acid-reactive substances in eye tissues. This resistance may be due to the higher ommochrome content. Accepted: 8 December 1998