Conversion of polychlorophenols by laccases with 1-hydroxybenzotriazole as a mediator

The action of purified laccase from the basidial fungi Cerrena unicolor and Trametes sp. on 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP) was studied, including reactions involving I-hydroxybenzotriazole as a mediator. Oxidation of 2,4,6-TCP by laccase without the mediator yielded 2,6-dichlorobenzoquinone as a primary conversion product, whereas PCP was not oxidized. Products of further conversion of 2,4,6-TCP and PCP formed with the presence of the mediator.     

Oxidation of 1-hydroxybenzotriazole by laccase and lignin peroxidase

A method to measure laccase and lignin peroxidase (LiP) activity at 408 nm (402–410 nm) using 1-hydroxybenzotriazole (HBT) was developed. The assay can be performed either as a kinetic measurement or as a stopped reaction using 5 mM Na-azide which improves the spectrum. Only white-rot fungal laccases and LiP were found to oxidize HBT to give shoulders or peaks at 402-410 nm. Phanerochaete and Phlebia manganese peroxidases did not give absorbance increase at 402–410 nm. © Rapid Science Ltd. 1998     

Role of 1-hydroxybenzotriazole in oxidation by laccase from Trametes versicolor. Kinetic analysis of the laccase-1-hydroxybenzotriazole couple

In the current studies, we used Lineweaver-Burke analysis to examine the role of 1-hydroxybenzotriazole (HBT) in the oxidation of various compounds by laccase from Trametes versicolor. At low concentrations, HBT was a competitive inhibitor of the oxidation, but at high concentrations, it was a noncompetitive inhibitor. Analysis of the oxidation of ferrocytochrome c by the laccase-HBT couple showed that increasing the concentration of ferrocytochrome c did not affect the V(max) but reduced the apparent K(m). In addition, in the manganese peroxidase-Mn(II) reaction, which is a typical oxidation system by mediator, the apparent K(m) and V(max) increased as the concentration of the substrate 2,6-dimethoxyphenol was increased. These results indicate that HBT is involved in the binding of laccase and substrates that laccase cannot oxidize alone.     

Degradation of nonphenolic lignin by the laccase/1-hydroxybenzotriazole system

Phenolic and nonphenolic (permethylated) synthetic [14C]lignins were depolymerized by Trametes villosa laccase in the presence of a radical mediator, 1-hydroxybenzotriazole (HOBT). Gel permeation chromatography of the treated lignins showed that approximately 10% of their substructures were cleaved. The system also cleaved a beta-O-4-linked model compound, 1-(4-ethoxy-3-methoxy-ring-[14C]phenyl)-2-(2-methoxyphenoxy)-propane- 1,3-diol, and a beta-1-linked model, 1, 2-bis-(3-methoxy-4-[14C]methoxyphenyl)-propane-1,3-diol, that represent nonphenolic substructures in lignin. High performance liquid chromatography of products from the oxidized models showed that they were produced in sufficient yields to account for the ability of laccase/HOBT to depolymerize nonphenolic lignin.     

Hypothalamic huntingtin-associated protein 1 as a mediator of feeding behavior

The hypothalamus responds to circulating leptin and insulin in the control of food intake and body weight. A number of neurotransmitters in the hypothalamus, including gamma-aminobutyric acid (GABA), also have key roles in feeding. Huntingtin-associated protein 1 (Hap1) is expressed more abundantly in the hypothalamus than in other brain regions, and lack of Hap1 in mice leads to early postnatal death. Hap1 is also involved in intracellular trafficking of the GABA(A) receptor. Here, we report that fasting upregulates the expression of Hap1 in the rodent hypothalamus, whereas intracerebroventricular administration of insulin downregulates Hap1 by increasing its degradation through ubiquitination. Decreasing the expression of mouse hypothalamic Hap1 by siRNA reduces the level and activity of hypothalamic GABA(A) receptors and causes a decrease in food intake and body weight. These findings provide evidence linking hypothalamic Hap1 to GABA in the stimulation of feeding and suggest that this mechanism is involved in the feeding-inhibitory actions of insulin in the brain.     

Treatment of tetracycline antibiotics by laccase in the presence of 1-hydroxybenzotriazole

Tetracycline antibiotics are widely used in human and veterinary medicine; however, residual amounts of these antibiotics in the environment are of concern since they could contribute to selection of resistant bacteria. In this study, tetracycline (TC), chlortetracycline (CTC), doxycycline (DC) and oxytetracycline (OTC) were treated with laccase from the white rot fungus Trametes versicolor in the presence of the redox mediator 1-hydroxybenzotriazole (HBT). High performance liquid chromatography demonstrated that DC and CTC were completely eliminated after 15 min, while TC and CTC were eliminated after 1 h. This system also resulted in a complete loss of inhibition of growth of Escherichia coli and Bacillus subtilis and the green alga Pseudokirchneriella subcapitata with decreasing tetracycline antibiotic concentration. These results suggest that the laccase-HBT system is effective in eliminating tetracycline antibiotics and removing their ecotoxicity.     

Macrophage cytotoxicity: interleukin 1 as a mediator of tumor cytostasis

Purified macrophage interleukin 1 (IL 1) induced a concentration-dependent inhibition of the proliferation of two commonly used tumor cell target lines, the human myeloid K562 and the murine T lymphoma Eb. In contrast, mastocytoma-derived P815 cells were not inhibited. The cytostatic action of IL 1 was not associated with direct cytotoxicity and was only partially reversible. PGE or interferon did not appear to mediate these effects. IL 1 treatment of the multipotential K562 cells revealed no morphologic evidence for the induction of specific differentiation. FACS analysis of IL 1-treated K562 cells showed a rapid decrease in transferrin receptor density, and a more delayed, but highly significant, increase in HLA-A,B,C antigen density. These findings provide one explanation for the frequently reported macrophage cytostatic actions against tumor cells, and indicate as well that IL 1, like interferon, may enhance the expression of Class I MHC antigens. These observations further extend the range of IL 1 actions and underscore the fundamental and direct role of this monokine in macrophage antitumor activity.     

Utilization of 1-hydroxybenzotriazole in mixed anhydride coupling reactions

The coupling of Boc-Val-OH to either H-Pro-OBzl or H-Pro-Gly-Val-Gly-OBzl by the mixed anhydride method leads to the formation of a urethane by-product in yields of 40-60%. This side reaction can be suppressed by the addition of HOBt to the reaction mixture before the amino component is added. This results in a substantially increased yield of the desired peptide.     

Sphingosine-1-phosphate as a mediator involved in development of fibrotic diseases

Fibrosis is a pathological process characterized by massive deposition of extracellular matrix (ECM) such as type I/III collagens and fibronectin that are secreted by an expanded pool of myofibroblasts, which are phenotypically altered fibroblasts with more contractile, proliferative, migratory and secretory activities. Fibrosis occurs in various organs including the lung, heart, liver and kidney, resulting in loss of normal tissue architecture and functions. Myofibroblasts could originate from multiple sources including tissue-resident fibroblasts, epithelial and endothelial cells through mechanisms of epithelial/endothelial-mesenchymal transition (EMT/EndMT), and bone marrow-derived circulating progenitors called fibrocytes. Emerging evidence in recent years shows that sphingosine-1-phosphate (S1P) acts on several types of target cells and is engaged in pro-fibrotic inflammatory process and fibrogenic process through multiple mechanisms, which include vascular permeability change, leukocyte infiltration, and migration, proliferation and myofibroblast differentiation of fibroblasts. Many of these S1P actions are receptor subtype-specific. In these actions, S1P has multiple cross-talks with other cytokines, particularly transforming growth factor-β (TGFβ), which plays a major role in fibrosis. The cross-talks include the regulation of S1P production through altered expression and activity of sphingosine kinases in fibrotic lesions, altered expression of S1P receptors, and S1P receptor-mediated transactivation of TGFβ signaling pathway. These cross-talks may give rise to a feed-forward, amplifying loop between S1P and TGFβ, and possibly with other cytokines in stimulating fibrogenesis. Another lysophospholipid mediator lysophosphatidic acid has also been recently implicated in fibrosis. The lysophospholipid signaling pathways represent novel, promising therapeutic targets for treating refractory fibrotic diseases. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Ubiquitin depletion as a key mediator of toxicity by translational inhibitors

Cycloheximide acts at the large subunit of the ribosome to inhibit translation. Here we report that ubiquitin levels are critical for the survival of Saccharomyces cerevisiae cells in the presence of cycloheximide: ubiquitin overexpression confers resistance to cycloheximide, while a reduced ubiquitin level confers sensitivity. Consistent with these findings, ubiquitin is unstable in yeast (t(1/2) = 2 h) and is rapidly depleted upon cycloheximide treatment. Cycloheximide does not noticeably enhance ubiquitin turnover, but serves principally to block ubiquitin synthesis. Cycloheximide also induces UBI4, the polyubiquitin gene. The cycloheximide-resistant phenotype of ubiquitin overexpressors is also characteristic of partial-loss-of-function proteasome mutants. Ubiquitin is stabilized in these mutants, which may account for their cycloheximide resistance. Previous studies have reported that ubiquitin is destabilized in the absence of Ubp6, a proteasome-associated deubiquitinating enzyme, and that ubp6 mutants are hypersensitive to cycloheximide. Consistent with the model that cycloheximide-treated cells are ubiquitin deficient, the cycloheximide sensitivity of ubp6 mutants can be rescued either by ubiquitin overexpression or by mutations in proteasome subunit genes. These results also show that ubiquitin wasting in ubp6 mutants is proteasome mediated. Ubiquitin overexpression rescued cells from additional translational inhibitors such as anisomycin and hygromycin B, suggesting that ubiquitin depletion may constitute a widespread mechanism for the toxicity of translational inhibitors.     

Utilization of 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide (EDCI)/1-hydroxybenzotriazole (HOBt) as a polymerizing agent

The commonly used coupling reagents in peptide synthesessuch as dicyclohexylcabodiimide, diisopropylcarbodiimide and3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with or without1-hydroxybenzotriazole or N-hydroxysuccinimide have been used as polymerizing agents in the synthesis of elastic/plastic protein-based polymers. It was found that 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with 1-hydroxybenzotriazole gave equally good polymers comparable toconventional p-nitrophenol approach. Further, we present here the polymerization and characterization of structural andfunctional properties of poly(Val-Pro-Gly-Val-Gly), which is themost striking repeating sequence in the bovine and porcine elastins. The polymers obtained by both p-nitrophenol and 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide approach werecharacterized by carbon-13 and proton nuclear magnetic resonancespectroscopy, differential scanning calorimetry, circular dichroism and fourier transform infrared spectroscopy. These results conclude that poly(Val-Pro-Gly-Val-Gly) obtained by bothmethods were identical in all respects of physical and chemicalproperties indicates that 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with 1-hydroxybenzotriazole method can be conveniently employed to synthesize these polymers.     

Utilization of 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide (EDCI)/1-hydroxybenzotriazole (HOBt) as a polymerizing agent

Summary The commonly used coupling reagents in peptide syntheses such as dicyclohexylcabodiimide, diisopropyl-carbodiimide and 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with or without 1-hydroxybenzotriazole or N-hydroxysuccinimide have been used as polymerizing agents in the synthesis of elastic/plastic protein-based polymers. It was found that 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with 1-hydroxybenzotriazole gave equally good polymers comparable to conventionalp-nitrophenol approach. Further, we present here the polymerization and characterization of structural and functional properties of poly(Val-Pro-Gly-Val-Gly), which is the most striking repeating sequence in the bovine and porcine elastins. The polymers obtained by bothp-nitrophenol and 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide approach were characterized by carbon-13 and proton nuclear magnetic resonance spectroscopy, differential scanning calorimetry, circular dichroism and fourier transform infrared spectroscopy. These results conclude that poly(Val-Pro-Gly-Val-Gly) obtained by both methods were identical in all respects of physical and chemical properties indicates that 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with 1-hydroxybenzotriazole method can be conveniently employed to synthesize these polymers.     

Chromatin structure as a mediator of aging

The aging process is characterized by gradual changes to an organism’s macromolecules, which negatively impacts biological processes. The complex macromolecular structure of chromatin regulates all nuclear processes requiring access to the DNA sequence. As such, maintenance of chromatin structure is an integral component to deter premature aging. In this review, we describe current research that links aging to chromatin structure. Histone modifications influence chromatin compaction and gene expression and undergo many changes during aging. Histone protein levels also decline during aging, dramatically affecting chromatin structure. Excitingly, lifespan can be extended by manipulations that reverse the age-dependent changes to chromatin structure, indicating the pivotal role chromatin structure plays during aging.