A possible metabolic role foro-diphenoloxidase inMycobacterium leprae

Summary Among mycobacteria,Mycobacterium leprae is unique in its ability to oxidize a variety of diphenols to quinones in vitro. What physiologic roleo-diphenoloxidase has in the organism remained unknown. Reducing substrates like NADPH, NADH and ascorbic acid reacted with the quinone formed from dopa (3,4-dihydroxyphenylalanine); the substrates were oxidized and the quinone was reduced back to diphenol in the process. Since the quinone undergoes reversible oxidation-reduction, diphenoloxidase might serve as an alternative respiratory mechanism inM. leprae for the utilization of other substrates, as has been reported in plants.     

A possible method for improving the efficacy of dapsone

The antileprosy drug dapsone is unable to penetrate intact Mycobacterium leprae in vitro, as determined by its effect on o-diphenoloxidase in the bacilli. When combined with the peptide polylysine, the sulfone drug passes through the bacterial cell membranes, and penetrates the enzyme protein, resulting in a 100% inhibition of its activity.     

Medium- and short-chain dehydrogenase/reductase gene and protein families

The MDR superfamily with ~350-residue subunits contains the classical liver alcohol dehydrogenase (ADH), quinone reductase, leukotriene B4 dehydrogenase and many more forms. ADH is a dimeric zinc metalloprotein and occurs as five different classes in humans, resulting from gene duplications during vertebrate evolution, the first one traced to ~500 MYA (million years ago) from an ancestral formaldehyde dehydrogenase line. Like many duplications at that time, it correlates with enzymogenesis of new activities, contributing to conditions for emergence of vertebrate land life from osseous fish. The speed of changes correlates with function, as do differential evolutionary patterns in separate segments. Subsequent recognitions now define at least 40 human MDR members in the Uniprot database (corresponding to 25 genes when excluding close homologues), and in all species at least 10888 entries. Overall, variability is large, but like for many dehydrogenases, subdivided into constant and variable forms, corresponding to household and emerging enzyme activities, respectively. This review covers basic facts and describes eight large MDR families and nine smaller families. Combined, they have specific substrates in metabolic pathways, some with wide substrate specificity, and several with little known functions.     

Kinetic and structural evidence of the alkenal/one reductase specificity of human ζ-crystallin

Human ζ-crystallin is a Zn²⁺-lacking medium-chain dehydrogenase/reductase (MDR) included in the quinone oxidoreductase (QOR) family because of its activity with quinones. In the present work a novel enzymatic activity was characterized: the double bond α,β-hydrogenation of medium-chain 2-alkenals and 3-alkenones. The enzyme is especially active with lipid peroxidation products such as 4-hydroxyhexenal, and a role in their detoxification is discussed. This specificity is novel in the QOR family, and it is similar to that described in the distantly related alkenal/one reductase family. Moreover, we report the X-ray structure of ζ-crystallin, which represents the first structure solved for a tetrameric Zn²⁺-lacking MDR, and which allowed the identification of the active-site lining residues. Docking simulations suggest a role for Tyr53 and Tyr59 in catalysis. The kinetics of Tyr53Phe and Tyr59Phe mutants support the implication of Tyr53 in binding/catalysis of alkenal/one substrates, while Tyr59 is involved in the recognition of 4-OH-alkenals.     

环境微生物的分子生物学研究方法

本文介绍了在环境微生物生态研究中的分子生物学方法如分子杂交，聚合链式扩增技术PC震，rRNA基因同源分析法，新型凝胶电泳技术，生物醌谱图法等和应用。这些技术的使用将大大扩展微生物生态学研究的空间，并使得在分子水平研究生态问题的机制成为可能。     

Antifeedant nature of the quinone primin and its quinol miconidin fromMiconia spp

Summary The quinone primin (1) and its quinol miconidin (2) which occur naturally in Miconia spp. (Melastomataceae), were synthesized and then tested as potential antifeedants against 6 insect species. Antifeedant activity was found in all cases, ranging from primin (1) being most active againstPieris brassicae, to miconidin (2) being only slightly effective againstHeliothis armigera.     

Effects of isoxazolyl-naphthoquinoneimines on growth and oxygen radical production in Trypanosoma cruzi and Crithidia fasciculata

Several 4-(aminomethylisoxazolyl)-1,2-naphthoquinones inhibited growth and DNA synthesis in Trypanosoma cruzi and stimulated O2 uptake and O2-. generation by the parasite epimastigotes and their mitochondrial and microsomal membranes; these results support the idea that oxygen radicals play a role in quinone toxicity. Maximal effects on respiration and O2-. generation were observed with antimycin-inhibited cells. Similar results as well as stimulation of H2O2 production were obtained with Crithidia fasciculata despite the presence of catalase in this organism.     

Sur un mécanisme possible de la phosphorylation oxydative

Summary Two possible mechanisms for oxidative phosphorylation are suggested, based on participation of quinones in the process.Both of them postulate the 1–4 addition of inorganic phosphate on a reactive quinone isomer (the quinone-methide II) without exchange of the quinone oxygen atoms. They also account for the P₁-¹⁸OH₂ exchange observed during oxidative phosphorylation.

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Communication présentée au Symposium International de Chimie organique des Produits Naturels, Bruxelles (12–15 juin 1962). Résumé dans: Industrie Chimique Belge27, 558 (1962).     

Tailored substrates for studies of attached cell culture

Substrates for studies of the interactions of attached cells with extracellular matrix components are often prepared by allowing a protein to adsorb from solution onto a glass or polystyrene substrate. This method is simple and effective for many studies, but it can fail in cases that require rigorous control over the structure and composition of adsorbed protein. Self-assembled monolayers formed by the spontaneous ordering of terminally functionalized alkanethiols onto a gold substrate are a class of well-ordered substrates and provide a convenient method for tailoring substrates with ligands, proteins and other groups. Methods that can pattern the monolayers provide a general strategy to create substrates that control the size, shape and spacing of attached cells. This review illustrates recent work that has used these methods of surface chemistry to create tailored substrates for studies in cell biology. Received 14 November 1997; received after revision 10 March 1998; accepted 10 March 1998     

Gramicidin S and polymyxins: the revival of cationic cyclic peptide antibiotics

Gramicidin S and polymyxins are small cationic cyclic peptides and act as potent antibiotics against Gram-negative and Gram-positive bacteria by perturbing integrity of the bacterial membranes. Screening of a natural antibiotics library with bacterial membrane vesicles identified gramicidin S as an inhibitor of cytochrome bd quinol oxidase and an alternative NADH dehydrogenase (NDH-2) and polymyxin B as an inhibitor of NDH-2 and malate: quinone oxidoreductase. Our studies showed that cationic cyclic peptide antibiotics have novel molecular targets in the membrane and interfere ligand binding on the hydrophobic surface of enzymes. Improvement of the toxicity and optimization of the structures and clinical uses are urgently needed for their effective application in combating drug-resistant bacteria.     

Sclerotization of mosquito cuticle

The mode of sclerotization of Aedes aegypti pupal and adult cuticle was examined by employing biochemical and radioactive techniques. During larval-pupal metamorphosis, tyrosine is converted to tanning precursors and is incorporated into aryl-amino adducts and beta-crosslinks. The major hydrolysis product of beta-crosslinks in pupal cases is identified to be arterenone. Examination of tanning modes in five different mosquito species shows that the ratio of quinone to beta-sclerotization not only differs within the life stages of the insects, but also differs between species.     

A model of enzymatic kinetics]

In this Note, we study a system of differential equations representing the kinetics of an enzymatic reaction. For a closed system, and in the domain where it has a biological significance, it is shown that: (i) there exists a unique equilibrium point, which is an asymptotically stable point; (ii) if several enzymes act on the same substrates, the equilibrium values of the substrates concentrations take their values within the range of the equilibrium values achieved with each of these enzymes, (iii) the kinetics of the reaction can be approached by a single first-order differential equation, which may be seen as a generalization of the Michaelis equation.     

Essais d'élaboration des voies d'approche à la chimiothérapie du cancer. Action sur l'eau d'hydratation des tissus néoplasiques; action sur les protéines

Summary The recent successes in chemotherapy suggest that a solution of the same order should now be possible for cancer. The cause of cancer being still unknown, the therapeutic action aimed at is the creation in the organism of conditions unfavorable for cell multiplication. Certain of the factors essential for cell growth, such as the hydrocarbons, have already been studied in this light. The author has demonstrated the possibility of influencing the high degree of hydration of tumor tissue by the use of substances capable of effecting a homogeneous distribution of the dispersed substances in the aqueous phase. Furthermore, it was proposed to influence the protein repair accompanying cell multiplication by means of substances having an affinity for proteins. This was done to effect a condensation of protein elements before they reach the tumor, thus producing a return to the normal concentration of the free protein fraction, and, at the same time, a decrease in the turn-over of these elements through the tumor. Electrophoretic measurements made with theTiselius apparatus have made it possible to follow protein affinity and to suggest the synthesis of compounds containing quinone and imine groups which may prove valuable in future investigations along these lines.     

Enzyme–substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases

Protein ubiquitylation is an important post-translational modification, regulating aspects of virtually every biochemical pathway in eukaryotic cells. Hundreds of enzymes participate in the conjugation and deconjugation of ubiquitin, as well as the recognition, signaling functions, and degradation of ubiquitylated proteins. Regulation of ubiquitylation is most commonly at the level of recognition of substrates by E3 ubiquitin ligases. Characterization of the network of E3–substrate relationships is a major goal and challenge in the field, as this expected to yield fundamental biological insights and opportunities for drug development. There has been remarkable success in identifying substrates for some E3 ligases, in many instances using the standard protein–protein interaction techniques (e.g., two-hybrid screens and co-immunoprecipitations paired with mass spectrometry). However, some E3s have remained refractory to characterization, while others have simply not yet been studied due to the sheer number and diversity of E3s. This review will discuss the range of tools and techniques that can be used for substrate profiling of E3 ligases.     

Sumoylation regulates diverse biological processes

Ten years after its discovery, the small ubiquitin-like protein modifier (SUMO) has emerged as a key regulator of proteins. While early studies indicated that sumoylation takes place mainly in the nucleus, an increasing number of non-nuclear substrates have recently been identified, suggesting a wider stage for sumoylation in the cell. Unlike ubiquitylation, which primarily targets a substrate for degradation, sumoylation regulates a substrate’s functions mainly by altering the intracellular localization, protein-protein interactions or other types of post-translational modifications. These changes in turn affect gene expression, genomic and chromosomal stability and integrity, and signal transduction. Sumoylation is counter-balanced by desumoylation, and well-balanced sumoylation is essential for normal cellular behaviors. Loss of the balance has been associated with a number of diseases. This paper reviews recent progress in the study of SUMO pathways, substrates, and cellular functions and highlights important findings that have accelerated advances in this study field and link sumoylation to human diseases. Received 19 March 2007; received after version 16 July 2007; accepted 1 August 2007     

Role of extracellular matrix molecules in the development of the sodium current in quail mesencephalic neural crest cells

The occurrence of the voltage-dependent sodium current has been studied in developing neurons from quail mesencephalic neural crest on different substrates, using the whole-cell patch clamp technique. Explants from 9–12 somite embryos were cultured on dishes coated with type I collagen, fibronectin, laminin or on plastic dishes in a chemically defined medium. After 18 h of culture the sodium current was observed in 70% of the neurons tested, and at 24 h some of these neurons were able to generate an action potential. After 18–25 h cells grown on fibronectinor collagen I-coated dishes showed a significantly higher occurrence of the sodium current (83% and 84% respectively) as compared to cells grown on uncoated plastic dishes (51%). Moreover, in the presence of fibronectin, the current density of the sodium current was more than doubled in comparison with cells grown on other substrates.     

Hemmungswirkung verschiedener Chinone auf die Eiweißspaltung durch Papain

Summary With the intention to clarify the mechanism of action of the antibiotic effect of some quinones the inhibition of the activity of papain by various quinones was measured. The results let it appear improbable that there is any relation between the antibacterial effect of the quinones and their inhibition power on papain. On the other hand a close parallelism was found in the strength of inhibition of the same quinones on papain, urease and catalase. The mechanism of action of the quinone inhibition of these three enzymes must be regarded as a very similar one.

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Ausführliche Veröffentlichung mit weiteren Belegen folgt in den Monatsheften für Chemie (Wien).     

Novel roles for insulin receptor (IR) in adipocytes and skeletal muscle cells via new and unexpected substrates

The insulin signaling pathway regulates whole-body glucose homeostasis by transducing extracellular signals from the insulin receptor (IR) to downstream intracellular targets, thus coordinating a multitude of biological functions. Dysregulation of IR or its signal transduction is associated with insulin resistance, which may culminate in type 2 diabetes. Following initial stimulation of IR, insulin signaling diverges into different pathways, activating multiple substrates that have roles in various metabolic and cellular processes. The integration of multiple pathways arising from IR activation continues to expand as new IR substrates are identified and characterized. Accordingly, our review will focus on roles for IR substrates as they pertain to three primary areas: metabolism/glucose uptake, mitogenesis/growth, and aging/longevity. While IR functions in a seemingly pleiotropic manner in many cell types, through these three main roles in fat and skeletal muscle cells, IR multi-tasks to regulate whole-body glucose homeostasis to impact healthspan and lifespan.     

The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin β and ADAM10

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)—the constitutive α-secretase—is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin^?/? mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.