Transient Kinetic Analysis of Hydrogen Sulfide Oxidation Catalyzed by Human Sulfide Quinone Oxidoreductase

The first step in the mitochondrial sulfide oxidation pathway is catalyzed by sulfide quinone oxidoreductase (SQR), which belongs to the family of flavoprotein disulfide oxidoreductases. During the catalytic cycle, the flavin cofactor is intermittently reduced by sulfide and oxidized by ubiquinone, linking H₂S oxidation to the electron transfer chain and to energy metabolism. Human SQR can use multiple thiophilic acceptors, including sulfide, sulfite, and glutathione, to form as products, hydrodisulfide, thiosulfate, and glutathione persulfide, respectively. In this study, we have used transient kinetics to examine the mechanism of the flavin reductive half-reaction and have determined the redox potential of the bound flavin to be −123 ± 7 mV. We observe formation of an unusually intense charge-transfer (CT) complex when the enzyme is exposed to sulfide and unexpectedly, when it is exposed to sulfite. In the canonical reaction, sulfide serves as the sulfur donor and sulfite serves as the acceptor, forming thiosulfate. We show that thiosulfate is also formed when sulfide is added to the sulfite-induced CT intermediate, representing a new mechanism for thiosulfate formation. The CT complex is formed at a kinetically competent rate by reaction with sulfide but not with sulfite. Our study indicates that sulfide addition to the active site disulfide is preferred under normal turnover conditions. However, under pathological conditions when sulfite concentrations are high, sulfite could compete with sulfide for addition to the active site disulfide, leading to attenuation of SQR activity and to an alternate route for thiosulfate formation.     

Presence of Nonlinear Excitations in DNA Structure and their Relationship to DNA Premelting and to Drug Intercalation

Abstract

We propose that collectively localized nonlinear excitations (solitons) exist in DNA structure. These arise as a consequence of an intrinsic nonlinear ribose inversion instability that results in a modulated β alternation in sugar puckering along the polymer backbone. In their bound state, soliton-antisoliton pairs contain β premelted core regions capable of undergoing breathing motions that facilitate drug intercalation. We call such bound state structures—β premeltons. The stability of a β premelton is expected to reflect the collective properties of extended DNA regions and to be sensitive to temperature, pH, ionic strength and other thermodynamic factors. Its tendency to localize at specific nucleotide base sequences may serve to initiate site-specific DNA premelting and melting. We suggest that β premeltons provide nucleation centers important for RNA polymerase-promoter recognition. Such nucleation centers could also correspond to nuclease hypersensitive sites.     

Five birds,one stone: Neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody

Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.     

Detection of bacteriophage tracers in bivalve molluscan shellfish

Procedures to enumerate commonly used bacteriophage tracers in bivalve molluscan shellfish were evaluated. Bacteriophages specific to Serratia marcescens, Escherichia coli and Enterobacter cloacae can be recovered from shellfish flesh by chloroform treatment of homogenates and subsequent clarification of samples by centrifugation. Bacteriophages were enumerated using a soft agar overlay technique. Hard clams appeared to release toxic compounds during homogenization which dramatically reduced counts of Ent. clocae bacteriophage.     

Phosphorylation within a specific domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro

We have investigated the functional significance of phosphoserine residues that lie in the L protein-binding domain between amino acids 213 and 247 of the phosphoprotein (NS) of vesicular stomatitis virus. A series of mutant NS proteins were made by cell-free translation of mRNAs transcribed from the cloned gene. Site-directed substitution of alanine for both serine 236 and serine 242 essentially abolished RNA synthesis catalyzed by the NS-L complex. Substitution of either of these serines reduced RNA synthesis by 75%. Serine 218 played no major role in RNA synthesis. Phosphorylation of NS by the L protein was abrogated by substitution of either serine 236 or serine 242. These results indicate that phosphorylation of serines 236 and 242 in the NS protein regulates its binding with the L protein and the N-RNA template and is essential for activation of viral RNA synthesis.     

New coumarins from Seseli sibiricum

Three new coumarins, sesebrin(7-methoxy-5-[3-methylbut-2-enyloxy]-8-[3-methyl-2,3-epoxybutyl] coumarin), sesebrinol(7-methoxy-5-[3-methylbut-2-enyloxy]-8-[3-methyl-2,3-dihydroxybutyl] coumarin) and sibiricol (7-methoxy-5-hydroxy-8-[3-methylbut-2-enyl] coumarin) have been isolated from the roots of Seseli sibiricum, in addition to sitosterol and 7 known coumarins, isobergapten, bergapten, phellopterin, sibiricin, coumurrayin, osthenol and meranzin hydrate.     

The turnover of basal lamina glycosaminoglycan correlates with epithelial morphogenesis

The mouse embryonic submandibular epithelium begins as a single bud from the floor of the mouth which, under the influence of its surrounding mesenchyme, grows and forms lobules that subsequently branch repetitively. The lobular morphology of the 13-day epithelium is maintained by its basal lamina which is a continuous layer on the interlobular clefts but is interrupted on the distal aspects of the lobules. The structural integrity of this lamina is dependent upon its glycosaminoglycan (GAG) which, by histochemistry, is more abundant on the interlobular clefts than on the distal lobules. We have investigated the basis for these regional differences in the lamina by examining the synthesis and degradation of total GAG as well as the accumulation and loss of laminar GAG during the morphogenesis of the 13-day gland. Autoradiography and histochemistry show that laminar GAG is rapidly turning over. Although it is relatively stable in the interlobular clefts, GAG is rapidly degraded on the distal lobules. This difference can account for the regional variation in basal laminar GAG accumulation. The results of incorporation kinetics and precursor pool specific activities of total epithelial GAG show that the rate of GAG synthesis is greater than its rate of degradation in the base of the lobules, which includes the interlobular clefts. In contrast, during morphogenesis, the rate of GAG degradation becomes greater than its rate of replacement in the distal lobules. The epithelial stalk appears to be in the steady state regarding GAG metabolism. We propose (a) that the rapid laminar GAG degradation on the distal lobules produces the interruptions in the lamina, allowing epithelial growth and expansion, and (b) that the metabolic stability of laminar GAG on the interlobular clefts maintains the integrity of this lamina which serves as a cellular constraint. The results are consistent with a model for epithelial morphogenesis in which the mesenchyme remodels the lamina, which in turn, dictates epithelial morphology. Regulation of basal lamina turnover may be a general mechanism for controlling the behavior of epithelial cell populations.