Electron paramagnetic resonance properties and oxidation-reduction potentials of the molybdenum,flavin, and iron-sulfur centers of chicken liver xanthine dehydrogenase

The oxidation-reduction potentials of the various prosthetic groups in the native and desulfo forms of chicken liver xanthine dehydrogenase, determined by potentiometric titration in 0.05 m potassium phosphate buffer, pH 7.8, are: Mo(VI)/Mo(V) (native), ?357 mV; Mo(VI)/Mo(V) (desulfo), ?397 mV; Mo(V)/Mo(IV) (native), ?337 mV; Mo(V)/Mo(IV) (desulfo), ?433 mV; FAD/FADH · ?345 mV; FADH · FADH₂, ? 377 mV; (Fe/S)I_ox/(Fe/S)I_red, ?280 mV; (Fe/S)II_ox/(Fe/S)II_red, ? 275 mV. Titration at pH 6.8 revealed that the Mo and FAD centers but not the Fe/S centers are in prototropic equilibrium. Spectroscopic studies on the native and deflavinated enzymes show that environment of the flavin in xanthine dehydrogenase differs from that in bovine milk xanthine oxidase.     

Molybdenum cofactor biosynthesis in Escherichia coli. Requirement of the chlB gene product for the formation of molybdopterin guanine dinucleotide.

The chlorate-resistant mutants of Escherichia coli are affected in the biosynthesis of the molybdenum cofactor and show pleiotropic loss of the activities of those enzymes which require the cofactor. The molybdenum cofactor in all molybdoenzymes other than nitrogenase is a complex of the metal with a unique pterin termed molybdopterin. The molybdenum cofactor in a number of E. coli enzymes has been shown to contain GMP in addition to the metal-molybdopterin complex, with the GMP appended in pyrophosphate linkage to the terminal phosphate ester on the molybdopterin side chain. In this paper, we have examined the biochemistry of the chlB mutant and show that the gene product of the chlB locus is essential for the addition of the GMP moiety to form molybdopterin guanine dinucleotide, a step which occurs late in the cofactor biosynthetic pathway in E. coli. Sensitive techniques were developed for the identification of fluorescent derivatives of molybdopterin and of molybdopterin guanine dinucleotide in extracts of E. coli cells. Wild type cells were shown to contain both molybdopterin and molybdopterin guanine dinucleotide, while cells of chlB mutants were found to contain elevated levels of molybdopterin but no detectable molybdopterin guanine dinucleotide.     

Flanking AT-rich tracts cause a structural distortion in Z-DNA in plasmids

The effect of neighboring AT-rich sequences on the right-handed B to left-handed Z transition was investigated in plasmids. The supercoil stabilized Z-DNA structure in (CG) tracts 36 and 40 base pairs (bp) in length revealed an unexpected conformational aberration at defined C residues proximal to one end (colL) when the inserts were bilaterally flanked by an 80% AT-rich segment (90 bp on one side and 331 bp on the other). The presence of the perturbed Z-conformation required (CG) stretches longer than 32 bp and bilateral flanking by the AT-rich tracts, since plasmids with the (CG) tracts unilaterally flanked had an orthodox Z-structure. The thermodynamics of the negative super-coil-induced transitions were influenced only slightly by the neighboring AT-rich regions. Hence, the nature of Z-conformations in plasmids is markedly influenced by intrinsic structural features of the (Pur-Pyr) tract and by seemingly modest changes in the properties of neighboring sequences over a distance of several helical turns.     

Mechanisms of inactivation of molybdoenzymes by cyanide

The reduced forms of xanthine oxidase, xanthine dehydrogenase, aldehyde oxidase, and sulfite oxidase are inactivated by cyanide. Following gel filtration to remove excess of reductant and cyanide, the isolated enzymes remain inactive. Thiocyanate, a product of inactivation of the oxidized forms of the xanthine- and aldehyde-oxidizing enzymes by cyanide, is not released during inactivation of the reduced enzymes. Studies with [14C]cyanide show that, while stoichiometric binding is required for the onset of inactivation, its continued binding is not essential to maintenance of the inactivated state. Electron paramagnetic resonance and absorption spectroscopic studies on the isolated inactivated enzymes show that prosthetic groups other than molybdenum are fully oxidized but that the molybdenum centers are modified. Reactivation is accomplished by incubation with suitable oxidants. Aerobic reactivation of inactive sulfite oxidase required only 1 eq of ferricyanide/active site. However, under rigorously anaerobic conditions, 3 to 4 mol of ferricyanide/active site were reduced, indicating that the molybdenum centers in the inactive enzyme had been reduced below the levels attained by the native enzyme during catalysis.     

A unique subgenomic species of adenovirus 2 DNA generated under high multiplicities of infection.

We have identified a novel subgenomic viral DNA in KB cells infected with adenovirus 2 (Ad2) under high multiplicities of infection. KB cells were infected with Ad2 at multiplicities of infection greater than 100 PFU/cell. 32P-labeled viral DNA was selectively extracted by a modification of the method of Hirt (8) from the infected cells and analyzed by electrophoresis on agarose gels. In addition to full-length DNA (33 to 23 x 10(6) daltons), a unique subgenomic DNA species of about 12 to 13% (2.6 x 10(6) daltons) of full-length DNA in size was found in the infected cells. This subgenomic DNA was found to be double stranded and was not packaged inside the virus particles. This DNA could be isolated in large amounts (30 to 50% of total viral DNA) from infected cells. When cleaved with restriction endonuclease KpnI, the subgenomic DNA yielded two fragments, each corresponding to about 6% and 7% of the full-length genome in size.     

Cytotoxicity of glucose analogues in V79 multicell spheroids

Summary 2-Deoxy-d-glucose (2DG) and 5-thio-d-glucose (5TG) are glucose antimetabolites that are known to be selectively toxic to hypoxic cells grown as single cells or as monolayer cultures. These analogues were toxic to Chinese hamster V79 cells grown as multicell spheroids even under aerobic conditions. When spheroids, 500- to 600-μm diameter, were exposed to 7.5mm of these chemicals for 3 days, the number of clonogenic cells per spheroid dropped to 50% for 5-thio-d-glucose and 20% for 2-deoxy-d-glucose, relative to control values. Survivals were reduced to less than 1% when the experiment was repeated in glucose-free medium. Scanning electron photomicrographs of spheroids treated with 7.5mm of either analogue showed extensive damage to the outer cells. The cell killing observed was much more than could be predicted on the basis of the hypoxic fraction known to be present in these spheroids. The crowded tumor-like environment may make the cells vulnerable to the cytotoxic action of glucose analogues and other glycolytic inhibitors. Supported by the Ontario Cancer Treatment and Research Foundation, London Clinic.     

Synthesis of 18-substituted steroids Part II (1). Improvements in the preparation of 18-hydroxyprogesterone.

18-Hydroxyprogesterone is conveniently prepared from 3beta-acetoxypregn-5-en-20beta-ol by a modified route. 3beta-Acetoxy-18-iodopregn-5-en-20-one, obtained by the hypoiodite-photolysis procedure and oxidation, is treated with methanolic silver acetate to give the 18, 20-epoxy-20-methoxy derivative, which crystallises directly without need for chromatography. Hydrolysis of the 3-acetate, and a modified Oppenauer oxidation, gave 18-hydroxy-progesterone in 24% over-all yield.     

Inflammatory Response to Fine Particulate Air Pollution Exposure: Neutrophil versus Monocyte

Objectives

Studies have shown that chronic exposure to ambient fine particulate matter (less than 2.5 µm in aerodynamic diameter, PM_2.5) pollution induces insulin resistance through alterations in inflammatory pathways. It is critical to study how the immune system responds to this stimulant, which has been linked to cardiovascular and autoimmune diseases, but few studies have been focused on such involvement of both neutrophils and monocytes in a timely manner. We hypothesized that the neutrophil was involved in the inflammatory response to air pollution.

Methods and Results

C57BL/6 mice were exposed to PM_2.5 or filtered air (6 hours/day, 5 days/week) for 5, 14, and 21 days, respectively, in Columbus, OH. At the end of each of the exposure periods, we investigated the inflammatory response through flow cytometry, histology, intravital microscopy, and real-time PCR. PM_2.5-exposed mice demonstrated a significant inflammatory response after 5 days of exposure. In the lung tissue and bronchoalveolar lavage fluid, monocytes/macrophages showed a transient response, while neutrophils showed a cumulative response. In addition, exposure to PM_2.5 resulted in elevation of the monocyte chemoattractant protein 1 (MCP-1) cytokine, a monocyte/macrophage attractant in blood, at an early stage of exposure.

Conclusions

These findings suggest that PM_2.5 exposure induces the inflammatory responses from both macrophages and neutrophils involvement.     

Safe Oral Triiodo-L-Thyronine Therapy Protects from Post-Infarct Cardiac Dysfunction and Arrhythmias without Cardiovascular Adverse Effects

Background

A large body of evidence suggests that thyroid hormones (THs) are beneficial for the treatment of cardiovascular disorders. We have shown that 3 days of triiodo-L-thyronine (T3) treatment in myocardial infarction (MI) rats increased left ventricular (LV) contractility and decreased myocyte apoptosis. However, no clinically translatable protocol is established for T3 treatment of ischemic heart disease. We hypothesized that low-dose oral T3 will offer safe therapeutic benefits in MI.

Methods and Results

Adult female rats underwent left coronary artery ligation or sham surgeries. T3 (~6 μg/kg/day) was available in drinking water ad libitum immediately following MI and continuing for 2 month(s) (mo). Compared to vehicle-treated MI, the oral T3-treated MI group at 2 mo had markedly improved anesthetized Magnetic Resonance Imaging-based LV ejection fraction and volumes without significant negative changes in heart rate, serum TH levels or heart weight, indicating safe therapy. Remarkably, T3 decreased the incidence of inducible atrial tachyarrhythmias by 88% and improved remodeling. These were accompanied by restoration of gene expression involving several key pathways including thyroid, ion channels, fibrosis, sympathetic, mitochondria and autophagy.

Conclusions

Low-dose oral T3 dramatically improved post-MI cardiac performance, decreased atrial arrhythmias and cardiac remodeling, and reversed many adverse changes in gene expression with no observable negative effects. This study also provides a safe and effective treatment/monitoring protocol that should readily translate to humans.