23761261
Interaction between Caveolin-1 and the Reductase Domain of Endothelial Nitric-oxide Synthase
Endothelial nitric-oxide synthase (eNOS) is targeted to caveoli through interaction with caveolin-1 (cav-1). cav-1 binding to a consensus site in the eNOS oxygenase domain is proposed to antagonize calmodulin (CaM) binding and thereby inhibit eNOS nitric oxide (NO) synthesis. To study the mechanism, we examined how cav-1 scaffolding domain peptide (amino acids 82–101; cav-1P) would affect NO synthesis, NADPH oxidation, cytochrome c reduction, and ferricyanide reduction by full-length eNOS or its isolated oxygenase and reductase domains. Cav-1P equivalently inhibited NO synthesis and NADPH oxidation by full-length eNOS in a manner reversible by CaM but did not affect NADPH-independent NO synthesis by full-length eNOS or its oxygenase domain, indicating inhibition required the reductase domain. Similar concentrations of cav-1P inhibited cytochromec reduction by full-length eNOS or the reductase domain (amino acids 492–1205) in a CaM-reversible manner, indicating cav-1P interaction with reductase or full-length eNOS are equivalent. Ferricyanide reduction was unaffected by cav-1P in all cases. Immunoblotting showed that full-length eNOS, eNOS oxygenase, and eNOS reductase all bound to an immobilized glutathioneS-transferase-cav-1 fusion protein. Thus, cav-1 interacts independently with both oxygenase and reductase domains of eNOS. The reductase interaction occurs independent of a cav-1 binding motif, is CaM-reversible, and is of sufficient affinity to match cav-1P inhibition of NO synthesis by full-length eNOS. We propose that cav-1 binding to eNOS reductase compromises its ability to bind CaM and to donate electrons to the eNOS heme, thereby inhibiting NO synthesis.
25660146
Volatilization of Mercury by Resting Mercury-Resistant Bacterial Cells

The mercuric ion reduction system encoded by the Hg2+ inducible mer operon confers bacterial resistance to mercuric ion. The mer A gene product which is a FAD-containing enzyme catalyzes the reduction of Hg2+ to volatile elemental mercury with the help of intracellular thiols and NADPH as a cofactor (Schottel 1974; Summers and Silver 1978; Fox and Walsh 1982; Misra 1992). Our earlier studies have shown that growing cells of different mercury-resistant bacteria reduce Hg2+ compounds to Hg(O) (Ray et al. 1989; Pahan et al. 1990a; Gachhui et al. 1989). We have also shown the effect of thiol compounds and flavins on mercury-degrading enzyme activities in mercury-resistant bacteria (Pahan et al. 1990b). Here we report that resting cells of mercury-resistant bacteria survive in a buffer system for several hours, synthesize inducible mercury-degrading enzymes and volatilize mercury from a mercury-containing buffer system. We know of no information regarding studies of mercury-degrading enzymes in resting mercury-resistant bacterial cells.
32191850
Mutational Analysis of the Tetrahydrobiopterin-binding Site in Inducible Nitric-oxide Synthase*
Inducible nitric-oxide synthase (iNOS) is a hemeprotein that requires tetrahydrobiopterin (H4B) for activity. The influence of H4B on iNOS structure-function is complex, and its exact role in nitric oxide (NO) synthesis is unknown. Crystal structures of the mouse iNOS oxygenase domain (iNOSox) revealed a unique H4B-binding site with a high degree of aromatic character located in the dimer interface and near the heme. Four conserved residues (Arg-375, Trp-455, Trp-457, and Phe-470) engage in hydrogen bonding or aromatic stacking interactions with the H4B ring. We utilized point mutagenesis to investigate how each residue modulates H4B function. All mutants contained heme ligated to Cys-194 indicating no deleterious effect on general protein structure. Ala mutants were monomers except for W457A and did not form a homodimer with excess H4B and Arg. However, they did form heterodimers when paired with a full-length iNOS subunit, and these were either fully or partially active regarding NO synthesis, indicating that preserving residue identities or aromatic character is not essential for H4B binding or activity. Aromatic substitution at Trp-455 or Trp-457 generated monomers that could dimerize with H4B and Arg. These mutants bound Arg and H4B with near normal affinity, but Arg could not displace heme-bound imidazole, and they had NO synthesis activities lower than wild-type in both homodimeric and heterodimeric settings. Aromatic substitution at Phe-470 had no significant effects. Together, our work shows how hydrogen bonding and aromatic stacking interactions of Arg-375, Trp-457, Trp-455, and Phe-470 influence iNOSox dimeric structure, heme environment, and NO synthesis and thus help modulate the multiple effects of H4B.
36570389
Elimination of Mercury and Organomercurials by Nitrogen-Fixing Bacteria
Bacteria isolated from mercury-polluted environments are often resistant to mercuric ions (Hg{sup 2+}) and organomercurials. Plasmids determining mercury resistance have been well characterized in gram-negative system. However, in Staphylococcus aureus mercury resistance has been found to be chromosomally determined. The known mechanism of bacterial Hg{sup 2+}-resistance is detoxification of the toxic Hg{sup 2+} by its enzymatic transformation by mercuric reductase to Hg (o). Organomercurial lyase mediates the degradation of organomercurial compounds to Hg{sup 2+}. Mercury and organomercurial resistances have been studied in different bacterial genera. There is little information on Hg-resistance in N{sub 2}-fixing soil bacteria, however, in many developing countries, including India, mercury pollution is still a problem because Hg-based pesticides and fungicides are still used routinely as seed-dressers in agriculture to control soil-borne and seed-borne fungal diseases. Volatilization of Hg from laboratory media by mercury-resistant bacteria containing low levels of mercury has been reported by several workers. It is interesting to note that N{sub 2}-fixing, Hg-resistant soil isolates could volatilize Hg from medium containing very high amounts of HgCl{sub 2}. In the present paper we report the volatilization patterns of five N{sub 2}-fixing bacterial strains, the effect of different inducers on mercuric reductase, and the pattern of substratemore » utilization by organomercurial lyase. In the presence of a low concentration of HgCl{sub 2}. enzymatic detoxification is sufficient to combat the adverse situation created by the presence of Hg{sup 2+} ions. In the presence of a high concentration of HgCl{sub 2}, intracellular sequestration by Hg{sup 2+} binding components may play an additional role in counteracting Hg-toxicity.« less
43633481
Interactions of Escherichia coli thioredoxin, the processivity factor, with bacteriophage T7 DNA polymerase and helicase.
Escherichia coli thioredoxin binds to a unique flexible loop of 71 amino acid residues, designated the thioredoxin binding domain (TBD), located in the thumb subdomain of bacteriophage T7 gene 5 DNA polymerase. The initial designation of thioredoxin as a processivity factor was premature. Rather it remodels the TBD for interaction with DNA and the other replication proteins. The binding of thioredoxin exposes a number of basic residues on the TBD that lie over the duplex region of the primer-template and increases the processivity of nucleotide polymerization. Two small solvent-exposed loops (loops A and B) located within TBD electrostatically interact with the acidic C-terminal tail of T7 gene 4 helicase-primase, an interaction that is enhanced by the binding of thioredoxin. Several basic residues on the surface of thioredoxin in the polymerase-thioredoxin complex lie in close proximity to the TBD. One of these residues, lysine 36, is located proximal to loop A. The substitution of glutamate for lysine has a dramatic effect on the binding of gene 4 helicase to a DNA polymerase-thioredoxin complex lacking charges on loop B; binding is decreased 15-fold relative to that observed with wild-type thioredoxin. This defective interaction impairs the ability of T7 DNA polymerase-thioredoxin together with T7 helicase to mediate strand displacement synthesis. This is the first demonstration that thioredoxin interacts with replication proteins other than T7 DNA polymerase.
20590744
A two-stage variable-stringency semiparametric method for mapping quantitative-trait loci with the use of genomewide-scan data on sib pairs.
Genomewide scans for mapping loci have proved to be extremely powerful and popular. We present a semiparametric method of mapping a quantitative-trait locus (QTL) or QTLs with the use of sib-pair data generated from a two-stage genomic scan. In a two-stage genomic scan, either the entire genome or a large portion of the genome is saturated with low-density markers at the first stage. At the second stage, the intervals that are identified as probable locations of the trait loci, by means of analysis of data from the first stage, are then saturated with higher-density markers. These data are then analyzed for fine mapping of the loci. Our statistical strategy for analysis of data from the first stage is a low-stringency method based on the rank correlation of squared trait-difference values of the sib pairs and the estimated identity-by-descent scores at the marker loci. We suggest the use of a low-stringency method at the first stage, to save on computational time and to avoid missing any marker interval that may contain the trait loci. For analysis of data from the second stage, we have developed a high-stringency nonparametric-regression approach, using the kernel-smoothing technique. Through extensive simulations, we show that this approach is more powerful than is a currently used method for mapping QTLs by use of sib pairs, particularly in the presence of dominance and epistatic effects at the trait loci.
37750469
Two modes of interaction of the single-stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase-thioredoxin complex.
The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD. gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions.
31255336
Abnormal radiological features in a multiple myeloma patient: a case report and radiological review of myelomas.
Multiple myeloma is the prototype of malignant monoclonal gammopathies. The most common skeletal sites are pelvis, skull, spine, ribs and femoral and humeral shafts. The classic radiographic presentation of multiple myeloma is lytic skeletal lesions. Other types of presentation include sclerotic and porotic changes. Primary sclerotic manifestations are rare and occur in only 3% of cases. Although exceptional, multiple myeloma must be borne in mind in the presence of bone sclerosis. This report presents a patient with multiple myeloma with a sunburst/hair-on-end pattern on the radiograph and sclerotic skeletal lesions.
6587208
Macrophage cholesteryl ester mobilization and atherosclerosis.
Accumulation of cholesteryl esters (CE) stored as cytoplasmic lipid droplets is the main characteristic of macrophage foam cells that are central to the development of atherosclerotic plaques. Since only unesterified or free cholesterol (FC) can be effluxed from the cells to extracellular cholesterol acceptors, hydrolysis of CE is the obligatory first step in CE mobilization from macrophages. This reaction, catalyzed by neutral cholesteryl ester hydrolase (CEH), is increasingly being recognized as the rate-limiting step in FC efflux. CEH, therefore, regulates the process of reverse cholesterol transport and ultimate elimination of cholesterol from the body. In this review, we summarize the earlier controversies surrounding the identity of CEH in macrophages, discuss the characteristics of the various candidates recognized to date and examine their role in mobilizing cellular CE and thus regulating atherogenesis. In addition, physiological requirements to hydrolyze lipid droplet-associated substrate and complexities of interfacial catalysis are also discussed to emphasize the importance of evaluating the biochemical characteristics of candidate enzymes that may be targeted in the future to attenuate atherosclerosis.