Roles of the threonine 407, aspartic acid 417, and threonine 419 residues in P450 2B1 in metabolism

We have previously observed that the quadruple (S407T-N417D-A419T-K473M) and triple (S407T-N17D-A419T) mutants of the chimeric construct of P450 2B1/2B2 do not undergo mechanism-based inactivation by 17alpha-ethynylestradiol (17EE) and tert-butyl 1-methyl-2-propynyl ether (tBMP). The ability of these mutants to metabolize 17EE, benzphetamine, and testosterone has been investigated. The profile for 17EE metabolism by both mutants was characteristic of both wild-types. The two mutants metabolized testosterone to form androstenedione with no formation of the hydroxy products as was seen with both the wild-types. Benzphetamine metabolism by the mutants showed that both mutants exhibited an increased tendency to catalyze demethylation rather than debenzylation. In the presence of the alternate oxidants cumene hydroperoxide and tert-butyl hydroperoxide, the wild-type 2B1 was not inactivated by 17EE. Metabolism of 17EE by 2B1 supported by these alternate oxidants revealed differences in the metabolites that may be related to the inability of 2B1 to be inactivated under these conditions.     

Iopamidol myelography-induced seizures

Iopamidol, a water-soluble contrast agent, has been rarely associated with seizures. We describe a case of generalized tonic-clonic seizure after cervical myelography with iopamidol in a previously healthy young man. In patients presenting with seizures, a history of recent myelography should be considered as an etiology. Iopamidol myelography may be associated with a risk of seizures. Clinicians need to be aware of this complication and inform their patients about such risk.     

Recombinant D. radiodurans cells for bioremediation of heavy metals from acidic/neutral aqueous wastes

The stability and superior metal bioremediation ability of genetically engineered Deinococcus radiodurans cells, expressing a non-specific acid phosphatase, PhoN in high radiation environment has already been established. The lyophilized recombinant DrPhoN cells retained PhoN activity and uranium precipitation ability. Such cells also displayed an extended shelf life of 6 months during storage at room temperature and showed surface associated precipitation of uranium as well as other metals like cadmium. Lyophilized cells, immobilized in polyacrylamide gels could be used for uranium bioprecipitation in a flow through system resulting in 70% removal from 1mM input uranium solution and a loading of 1 g uranium/g dry weight cells. Compared with a batch process which achieved a loading of 5.7 g uranium/g biomass, the efficiency of the column process was low due to clogging of the column by the precipitate.     

Signaling from the secretory granule to the nucleus

Neurons and endocrine cells use a complex array of signaling molecules to communicate with each other and with various targets. The majority of these signaling molecules are stored in specialized organelles awaiting release on demand: 40-60 nm vesicles carry conventional or small molecule neurotransmitters, and 200-400 nm granules contain bioactive peptides. The supply of small molecule neurotransmitters is tightly regulated by local feedback of synthetic rates and transport processes at sites of release. The larger granules that contain bioactive peptides present the secretory cell with special challenges, as the peptide precursors are inserted into the lumen of the secretory pathway in the cell soma and undergo biosynthetic processing while being transported to distant sites for eventual secretion. One solution to this dilemma in information handling has been to employ proteolytic cleavage of secretory granule membrane proteins to produce cytosolic fragments that can signal to the nucleus, affecting gene expression. The use of regulated intramembrane proteolysis to signal from secretory granules to the nucleus is compared to its much better understood role in relaying information from the endoplasmic reticulum by SREBP and ATF6 and from the plasma membrane by cadherins, Notch and ErbB4.     

Sialoglycosylation of RBC in Visceral Leishmaniasis Leads to Enhanced Oxidative Stress, Calpain-Induced Fragmentation of Spectrin and Hemolysis

Visceral leishmaniasis (VL) caused by the intracellular parasite Leishmania donovani accounts for an estimated 12 million cases of human infection. It is almost always associated with anemia, which severely complicates the disease course. However, the pathological processes leading to anemia in VL have thus far not been adequately characterized to date. In studying the glycosylation patterns of peripheral blood cells we found that the red blood cells (RBC) of VL patients (RBC(VL)) express eight 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) that are not detected in the RBC of healthy individuals (RBC(N)). At the same time, the patients had high titers of anti-9-O-AcSGP IgG antibodies in their sera. These two conditions appear to be linked and related to the anemic state of the patients, as exposure of RBC(VL) but not RBC(N) to anti-9-O-AcSGPs antibodies purified from patient sera triggered a series of responses. These included calcium influx via the P/Q-type but not L-type channels, activation of calpain I, proteolysis of spectrin, enhanced oxidative stress, lipid peroxidation, externalization of phosphatidyl serine with enhanced erythrophagocytosis, enhanced membrane fragility and, finally, hemolysis. Taken together, this study suggests that the enhanced hemolysis is linked to an impairment of membrane integrity in RBC(VL) which is mediated by ligand-specific interaction of surface 9-O-AcSGPs. This affords a potential explanation for the structural and functional features of RBC(VL) which are involved in the hemolysis related to the anemia which develops in VL patients.     

Reversible and time-dependent inhibition of the hepatic cytochrome P450 steroidal hydroxylases by the proestrogenic pesticide methoxychlor in rat and human

Methoxychlor, a currently used pesticide, is demethylated and hydroxylated by several hepatic microsomal cytochrome P450 enzymes. Also, methoxychlor undergoes metabolic activation, yielding a reactive intermediate (M*) that binds irreversibly and apparently covalently to microsomal proteins. The study investigated whether methoxychlor could inhibit or inactivate certain liver microsomal P450 enzymes. The regioselective and stereoselective hydrox-ylation of testosterone and the 2-hydroxylation of estradiol (E₂) were utilized as markers of the P450 enzymes inhibited by methoxychlor. Both reversible and time-dependent inhibition were examined. Coincubation of methoxychlor and testosterone with liver microsomes from phenobarbital treated (PB-microsomes) male rats, yielded marked diminution of 2α- and 16α-testosterone hydroxylation, indicating strong inhibition of P4502C11 (P450h). Methoxychlor moderately inhibited 2β-, 7α-, 15α-, 15β-, and 16β-hydroxylation and androstenedi-one formation. There was only a weak inhibition of 6β-ydroxylation of testosterone. The methox-ychlor-mediated inhibition of 6β-hydroxylation was competitive. By contrast, when methoxychlor was permitted to be metabolized by PB-microsomes or by liver microsomes from pregnenolone-16α-car-bonitrile treated rats (PCN-microsomes) prior to addition of testosterone, a pronounced time-dependent inhibition of 6β-hydroxylation was observed, suggesting that methoxychlor inactivates the P450 3A isozyme(s). The di-demethylated methoxychlor (bis-OH-M) and the tris-hydroxy (ca-techol) methoxychlor metabolite (tris-OH-M) inhibited 6β-hydroxylation in PB-microsomes competitively and noncompetitively, respectively; however, these methoxychlor metabolites did not exhibit a time-dependent inhibition. Methoxychlor inhibited competitively the formation of 7α-hydroxytestosterone (7α-OH-T) and 16α-hydroxy-testosterone (16α-OH-T) but exhibited little or no time-dependent inhibition of generation of these metabolites, indicating that P450s 2A1, 2B1/B2, and 2C11 were inhibited but not inactivated. Methoxychlor inhibited in a time-dependent fashion the 2-hydroxylation of E2 in PB-microsomes. However, bis-OH-M exhibited solely reversible inhibition of the 2-hydroxylation, supporting our conclusion that the inactivation of P450s does not involve participation of the demethylated metabolites. Both competitive inhibition and time-dependent inactivation of human liver P450 3A (6β-hydroxylase) by methoxychlor, was observed. As with rat liver microsomes, the human 6β-hydroxylase was inhibited by bis-OH-M and tris-OH-M competitively and noncompetitively, respectively. Testosterone and estradiol strongly inhibited the irreversible binding of methoxychlor to microsomal proteins. This might explain the “clean” competitive inhibition by methoxychlor of the 6β-OH-T formation when the compounds were coin-cubated. Glutathione (GSH) has been shown to interfere with the irreversible binding of methoxychlor to PB-microsomal proteins. The finding that the coincubation of GSH with methoxychlor partially diminishes the time-dependent inhibition of 6β-hydroxylation provides supportive evidence that the inactivation of P450 3A isozymes by methoxychlor is related to the formation of M*.     

Phenotypic consequences of transforming growth factor β1 gene ablation in murine embryonic fibroblasts: Autocrine control of cell proliferation and extracellular matrix biosynthesis

The profound effects of transforming growth factor β1 (TGF-β1) on the immune system, cardiogenesis, in yolk sac hematopoeisis and in differentiation of endothelium have been demonstrated by detailed analyses of TGF-β1 knockout mice during embryogenesis. We have systematically examined the autocrine and paracrine roles of TGF-β1 in cell proliferation and in its ability to modulate the gene expression of selected components of extracellular matrix (ECM) using embryonic fibroblasts from TGF-β1 null mice (TGF-β1^−/−). The rates of cell proliferation of embryonic fibroblasts from normal mice (TGF-β1^+/+) and TGF-β1 null mice were compared by cell counting, by ³H thymidine incorporation, and by measuring the fraction of cells in the G1, S, and G2/M phases of the cell cycle by fluorescent activated cell sorting (FACS). Concurrently, the expression of pro-α1(I) collagen, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) was also quantified by hybridization of total mRNA from TGF-β1^+/+ and TGF-β1^−/− embryonic fibroblasts. We report that TGF-β1^−/− cells proliferated at about twice the rate of TGF-β1^+/+ cells. Further, TGF-β1 null fibroblasts accumulated and synthesized lower constitutive levels of pro-α1(I) collagen, fibronectin, and PAI-1 mRNA. The quantitative differences in the rates of cell proliferation and ECM gene expression between TGF-β1^+/+ and TGF-β1^−/− cells could be eliminated by treatment of TGF-β1^+/+ cells with a neutralizing antibody of TGF-β1. Thus, our results are consistent with the hypothesis that TGF-β1 acts as a negative autocrine regulator of growth and a positive autocrine regulator of ECM biosynthesis in embryonic fibroblasts. 176:67–75, 1998. Published 1998 Wiley-Liss, Inc.

1 This article was prepared by a group of United States government employees and non-United States government employees, and as such is subject to 17 U.S.C. Sec. 105.

     

Identification of structural transitions in bacterial fatty acid binding proteins that permit ligand entry and exit at membranes

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein–FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a “closed” FakB1 state to an “open” state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior.     

A systematic review and integrative approach to decode the common molecular link between levodopa response and Parkinson’s disease

Background

PD is a progressive neurodegenerative disorder commonly treated by levodopa. The findings from genetic studies on adverse effects (ADRs) and levodopa efficacy are mostly inconclusive. Here, we aim to identify predictive genetic biomarkers for levodopa response (LR) and determine common molecular link with disease susceptibility. A systematic review for LR was conducted for ADR, and drug efficacy, independently. All included articles were assessed for methodological quality on 14 parameters. GWAS of PD were also reviewed. Protein-protein interaction (PPI) analysis using STRING and functional enrichment using WebGestalt was performed to explore the common link between LR and PD.

Results

From 37 candidate studies on levodopa toxicity, 18 genes were found associated, of which, CA_n STR 13, 14 (DRD2) was most significantly associated with dyskinesia, followed by rs1801133 (MTHFR) with hyper-homocysteinemia, and rs474559 (HOMER1) with hallucination. Similarly, 8 studies on efficacy resulted in 4 genes in which rs28363170, rs3836790 (SLC6A3) and rs4680 (COMT), were significant. To establish the molecular connection between LR with PD, we identified 35 genes significantly associated with PD. With 19 proteins associated with LR and 35 with PD, two independent PPI networks were constructed. Among the 67 nodes (263 edges) in LR, and 62 nodes (190 edges) in PD pathophysiology, UBC, SNCA, FYN, SRC, CAMK2A, and SLC6A3 were identified as common potential candidates.

Conclusion

Our study revealed the genetically significant polymorphism concerning the ADRs and levodopa efficacy. The six common genes may be used as predictive markers for therapy optimization and as putative drug target candidates.