Low Molecular Weight Mediators Research Articles

Production of Recombinant Laccase From Coprinopsis cinerea and Its Effect in Mediator Promoted Lignin Oxidation at Neutral pH.

Laccases are multi-copper oxidases that use molecular oxygen as the electron acceptor to oxidize phenolic and indirectly also non-phenolic substrates by mechanisms involving radicals. Due to their eco-friendliness and broad substrate specificity, laccases span a wide range of biotechnological applications. We have heterologously expressed a laccase from the coprophilic basidiomycete Coprinopsis cinerea (CcLcc9) in the methylotrophic yeast Pichia pastoris. The recombinant CcLcc9 (rCcLcc9) oxidized 2,6-dimethoxyphenol in the neutral pH range, and showed thermostability up to 70°C. The rCcLcc9 efficiently oxidized veratryl alcohol to veratraldehyde in the presence of low molecular weight mediators syringyl nitrile, methyl syringate and violuric acid, which are syringyl-type plant phenolics that have shown potential as natural co-oxidants for lignocellulosic materials. In addition, rCcLcc9 is able to depolymerize biorefinery hardwood lignin in the presence of methyl syringate and syringyl nitrile as indicated by gel permeation chromatography, and infrared spectral and nucleic magnetic resonance analyses. Furthermore, we showed that several added-value aromatic compounds, such as vanillin, vanillic acid, syringaldehyde, syringic acid and p-hydroxybenzoic acid, were formed during sequential biocatalytic chemical degradation of biorefinery lignin, indicating that rCcLcc9 harbors a great potential for sustainable processes of circular economy and modern biorefineries.

Enhancing catalytic functionality of Trametes versicolor IBL-04 laccase by immobilization on chitosan microspheres

Laccase/mediator systems are promising bio-remediating agents as the rates of reactions can be considerably enhanced in the presence of low molecular weight mediators. A monomeric 64-kDa laccase produced by Trametes versicolor IBL-04 was effectively immobilized onto chitosan microspheres using glutaraldehyde as activating/cross-linking agent. Chitosan beads developed from 2.5% (w/v) chitosan concentration and activated by 1.5% (v/v) glutaraldehyde solution displayed best laccase immobilization efficiency. Scanning electron microscopy showed that beads with encapsulated laccase on the surface were spherical in shape having large surface area. The chitosan beads immobilized laccase (denoted as CTS-Lac) exhibited a broader active pH and temperature range and showed maximum activity at pH 6.0 and 60°C. After immobilization, the affinity of the enzyme toward its substrate increased (Km decreased), leading to enhanced catalytic efficiency (Vmax increased). As compared to free laccase, the CTS-Lac showed significantly improved thermal and storage stability. The catalytic activity of the immobilized laccase was also demonstrated by the decolorization of five different textile reactive dyes. In the presence of 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as redox mediator, a maximum of 100% decolorization was achieved within the shortest time period of 4.0h, indicating the effectiveness of CTS-Lac for the removal of textile dyes from an aqueous solution. It also retained 80.19% of its activity after ten continuous decolorization cycles for Sandal-fix Red C4BLN. The kinetic, thermo-stability and dye-decolorizing characteristics of CTS-Lac/mediator system reflect its potential for use in industrial and environmental biotechnology.

PP073. Gestational hypertension: A study on placental expression of endothelial and inducible nitric oxide synthase and no metabolism

Hypertension is one of the most common medical disorders in pregnancy and a major cause of maternal and perinatal morbidity and death. In primates adequate development of the embryo, and later of the fetus, depends on a successful hemomonochorial placentation. Nitric oxide (NO) a low molecular weight mediator, induces vasodilatation, inhibits platelet aggregation, and prevents the adhesion of platelets to endothelial cells. Till date, no data are available regarding gestational hypertension (GH) placenta and no metabolism and related enzyme expression and activity. The present study aimed to evaluate eNOS and iNOS expression in the placentas of both normal and GH patients, by means of Real-Time quantitative PCR, measure placental nitric oxide and peroxynitrite levels in the same group of subjects, and correlate such findings with HELLP group already published. Fifteen patients with gestational hypertension and thirty healthy pregnant controls comparable for maternal and gestational age were enrolled in the study. Placental tissue was taken immediately after delivery. eNOS and iNOS mRNA levels were evaluated Real-Time quantitative PCR, whereas nitric oxide and peroxynitrite production was measured by a commercially available kit. Placental eNOS and iNOS mRNA levels were significantly reduced in GH (2,02-fold reduction and 2,33-fold reduction, respectively) when compared to controls. Conversely, NO and ONOO(-) production were significantly higher in GH group compared to control group (31.56±4.15nmol NO/mg prot vs. 23.98±5.14nmol NO/mg prot and 68.49±8.57 arbitrary fluorescence units vs 17.31±2.25 arbitrary fluorescence units; p<0, 05). Such results were compared to HELLP group obtained in an already published study. As from results herein reported, we can hypothesize that complex mechanisms involving NO pathways cause a placental vasculature damage. However, it is not easy to understand if these changes could be interpreted as causes or consequences of this pathologic state.

Investigation of the potential of microbial stripping of dyed cotton fabric using white rot fungi

This paper reports the results of a novel approach of investigating the potential of biochemical stripping of cotton fabric dyed with reactive black B dye using five indigenous strains of white rot fungi (WRF) in Kirk’s basal salts medium. The color stripping was measured in terms of color strength (K/S value) before and after treatment. Among the five strains of WRF, Ganoderma lucidum IBL-05 showed the best stripping (20.11%) in 15 days of incubation under continuous shaking conditions (120 rpm) at pH 4.5 and 35 °C. The biochemical stripping process using G. lucidum IBL-05 was developed by optimizing various parameters like temperature, pH, additional carbon and nitrogen sources, low molecular weight mediators and metal ions. Supplementation with additional carbon and nitrogen sources enhanced the stripping to 67.73% under optimum conditions of pH 4 and temperature 35 °C. Wheat bran (1 g/100 mL) and peptone (0.2 g/ 100 mL) were found to be the best carbon and nitrogen sources, respectively. The addition of MnSO4 (1 mM) as a mediator of ligninolytic enzymes and ZnSO4 (1 mM) as a metal ion activator, further enhanced the stripping to 90.59% under optimum conditions generating a transparent effluent at the end of 15 days.

Acute Exposure to Solar Simulated Ultraviolet Radiation Affects Oxidative Stress-Related Biomarkers in Skin, Liver and Blood of Hairless Mice

The ultraviolet (UV) region of solar radiation is a critical factor in the initiation and development of a number of skin diseases. However, it is not only skin which is directly exposed to solar light that is affected by UV radiation, through low molecular weight mediators, generated upon irradiation, "non-skin" tissues can also be affected. The aim of this study was to examine in detail, the acute effects of UVA and UVB wavebands on hairless mice. Female SKH-1 hairless mice were exposed to a single dose of UVB (200, 800 mJ/cm(2)) or UVA (10, 20 J/cm(2)) using a solar simulator. The effects on haematological parameters, activity and/or expression of antioxidant enzymes, level of glutathione (GSH), markers of oxidative damage (lipid peroxidation and carbonylated proteins) were analysed in erythrocytes, plasma, liver and whole skin homogenates. No macroscopic changes were observed either 4 or 24 h after UVA/UVB exposure. The blood count showed a significant increase in leukocyte number and reduction of platelets 4 h following UVA and UVB irradiation, which disappeared 24 h after irradiation except for the higher UVA dose. Changes in oxidative stress-related parameters, particularly activity of catalase (CAT) and superoxide dismutase (SOD) and level of GSH and lipid peroxidation products, were found in skin, erythrocytes and liver. The expression of several enzymes (CAT, SOD, glutathione transferase (GST), nicotinamide adenine dinucleotide (phosphate) quinone oxidoreductase (NQO1) and hem oxygenase-1 (HO-1)) in skin was affected following UVA and UVB radiation. Increase in carbonylated proteins was found in plasma and skin samples.

Proteomics as a Method for Early Detection of Cancer: A Review of Proteomics, Exhaled Breath Condensate, and Lung Cancer Screening

The study of expressed proteins in neoplasia is undergoing a revolution with the advent of proteomic analysis. Unlike genomic studies where individual changes may have no functional significance, protein expression is closely aligned with cellular activity. This perspective will review proteomics as a method of detecting markers of neoplasia with a particular emphasis on lung cancer and the potential to sample the lung by exhaled breath condensate (EBC). EBC collection is a simple, new, and noninvasive technique, which allows sampling of lower respiratory tract fluid. EBC enables the study of a wide variety of biological markers from low molecular weight mediators to macromolecules, such as proteins, in a range of pulmonary diseases. EBC may be applied to the detection of lung cancer where it could be a tool in early diagnosis. This perspective will explore the potential of applying proteomics to the EBC from lung cancer patients as an example of detecting potential biomarkers of disease and progression.

Effect of Fungal Laccase and Low Molecular Weight Mediators on Decolorization of Direct Blue Dye

This study was performed to examine the role of laccase from white-rot fungi, Cerrena unicolor and Trametes versicolor, with low molecular weight mediators, acetovanillone (AV) and acetosyringone (AS), in decolorizing of the textile dye, Chicago sky blue 6B (Direct Blue 1, DB1). The tested fungi were shown the ability to remove colour from DB1. The decolorizing activity of the dye was closely related to the lac- case from fungi. In the presence of AV or AS as co-substrate, decolorization was more extensive than that of the control. Moreover, at the presence of AS, the decolorization process was more effective than AV. The highly purified laccase of C. unicolor and T. versicolor also decolorized the dye. The addition of AV or AS enhanced this process.

Dysregulated Cytokine Metabolism, Altered Hepatic Methionine Metabolism and Proteasome Dysfunction in Alcoholic Liver Disease

Alcoholic liver disease (ALD) remains an important complication and cause of morbidity and mortality from alcohol abuse. Major developments in our understanding of the mechanisms of ALD over the past decade are now being translated into new forms of therapy for this disease process which currently has no FDA approved treatment. Cytokines are low molecular weight mediators of cellular communication, and the pro-inflammatory cytokine tumor necrosis factor (TNF) has been shown to play a pivotal role in the development of experimental ALD. Similarly, TNF levels are elevated in the serum of alcoholic hepatitis patients. Abnormal methionine metabolism is well documented in patients with ALD, with patients having elevated serum methionine levels, but low S-adenosylmethionine levels in the liver. On the other hand, S-adenosylhomocysteine and homocysteine levels are elevated in ALD. Recent studies have documented potential interactions between homocysteine and S-adenosylhomocysteine with TNF in the development of ALD. Altered proteasome function also is now well documented in ALD, and decreased proteasome function can cause hepatocyte apoptosis. Recently it has been shown that decreased proteasome function can also act synergistically to enhance TNF hepatotoxicity. Hepatocytes dying of proteasome dysfunction release pro-inflammatory cytokines such as Interleukin-8 to cause sustained inflammation. This article reviews the interactions of cytokines, altered methionine metabolism, and proteasome dysfunction in the development of ALD.

Electropolymerized flavin adenine dinucleotide as an advanced NADH transducer.

Electropolymerizing the prosthetic group (flavin adenine dinucleotide, FAD) responsible in the active sites of dehydrogenases for NAD(+)|NADH regeneration, we succeeded in mimicking enzyme activity. Poly(FAD) characterized by an additional polymer-type redox reaction has been discovered as a highly effective electrocatalyst for NADH oxidation: operating at the lowest potentials reported for NADH transducers (0.00 V, pH 7.4), poly(FAD) is characterized by the electrochemical rate constant of 1.8 +/- 0.6 x 10(-3) cm s(-1), which is at the level of the NADH mass-transfer constant. Flow injection analysis of NADH with the poly(FAD)-modified wall-jet electrode as a detector has been characterized by a linear calibration range prolonged down to 5 x 10(-7) M and a sensitivity of 0.08 A M(-1) cm(-2), which taking into account the dispersion coefficient ( approximately 3), is at the diffusion-limiting value. In contrast to the low molecular weight mediators able to exhibit similar electrocatalytic properties, poly(FAD)-modified electrodes are characterized by the dramatically improved stability and, thus, can be considered as the most advantageous NADH transducers for analytical chemistry.

Oxidation of a Tetrameric Nonphenolic Lignin Model Compound by Lignin Peroxidase

The present study maps the active site of lignin peroxidase in respect to substrate size using either fungal or recombinant wild type, as well as mutated, recombinant lignin peroxidases. A nonphenolic tetrameric lignin model was synthesized that contains beta-O-4 linkages. The fungal and recombinant wild type lignin peroxidase both oxidized the tetrameric model forming four products. The four products were identified by mass spectral analyses and compared with synthetic standards. They were identified as tetrameric, trimeric, dimeric, and monomeric carbonyl compounds. All four of these products were also formed from single turnover experiments. This indicates that lignin peroxidase is able to attack any of the C(alpha)-C(beta) linkages in the tetrameric compound and that the substrate-binding site is well exposed. Mutation of the recombinant lignin peroxidase (isozyme H8) in the heme access channel, which is relatively restricted and was previously proposed to be the veratryl alcohol-binding site (E146S), had little effect on the oxidation of the tetramer. In contrast, mutation of a Trp residue (W171S) in the alternate proposed substrate-binding site completely inhibited the oxidation of the tetrameric model. These results are consistent with lignin peroxidase having an exposed active site capable of directly interacting with the lignin polymer without the advent of low molecular weight mediators.

Activated mast cells produce interleukin 13.

When mast cells are activated through their immunoglobulin (Ig)E receptors, release of low molecular weight mediators like histamine is followed by secretion of multiple cytokines, including interleukin (IL)-3, IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor. Here we report that stimulated mast cells also synthesize IL-13 mRNA and protein; secretion of this cytokine may be of particular importance because of its ability to stimulate IgE expression. IL-13 transcripts detected by a semiquantitative reverse transcriptase-mediated polymerase chain reaction assay were induced within 30 min after stimulation of mast cells by dinitrophenyl plus monoclonal IgE anti-dinitrophenyl, and peaked at about 1 h. Within 3 h of IgE stimulation, secreted IL-13 bioactivity, estimated by proliferation of an IL-13-dependent cell line, reached levels equivalent to 1-2 ng/ml of IL-13. When added to human B lymphocytes, the mast cell-derived IL-13 activity (like bone fide IL-13) induced Ig C epsilon transcripts, DNA recombination characteristic of the isotype switch to C epsilon, and the secretion of IgE protein. These results suggest a model of local positive feedback interactions between mast cells and B cells, which could play a role in the pathogenesis of atopy.

Host responses in periodontal diseases: current concepts.

In periodontal diseases, bacteria trigger inflammatory host responses which, along with the direct destructive effects of the bacteria, cause most of the tissue destruction. Periodontal inflammatory responses are, by and large, immunologic, and our understanding of these reactions has been advanced by the explosion of knowledge in immunobiology, some of which is discussed in this review. Understanding the role of immune cells and their regulatory cell surface molecules such as the MHC, CD antigens, and receptors, as well as knowledge of effector systems set into motion such as phagocytes and cytotoxic T-cells, and the effector molecules such as antibodies, complement, and cytokines, have led to better understanding of the complex pathogenesis of periodontal disease. The role of mediators including the matrix metalloproteinases, proteoglycans, the kinins and anaphylatoxins, and low molecular weight mediators including products of arachidonic metabolism is beginning to be elucidated in periodontal disease. Important avenues of research for development of diagnostic tests based upon host response are apparent. For example, tissue products released during periodontal inflammation including the metalloproteinases, elastase, cytokines, prostaglandins, antibodies, and complement components may provide the basis for future diagnostic indicator tests. The recognition that the neutrophil/antibody/complement axis is critical for protection against periodontal bacteria and that abnormalities in this system often lead to increased periodontal susceptibility provide approaches for the development of diagnostic tests assessing risk. A group of factors which are negative regulators of inflammation including TGF-beta, gamma-interferon, and IL-1 receptor antagonist provide potential for assessment of periodontal disease in remission or in the healing phase. Finally, factors such as HLA associations and the molecular basis for neutrophil abnormalities may provide genetic markers for periodontal disease susceptibility. Diagnostic factors based upon host response measures offer great potential for predicting host susceptibility and will likely be used in combination with microbial diagnostics which identify specific infecting organisms.

Rat liver insulin mediator which stimulates pyruvate dehydrogenase phosphatase contains galactosamine and D-chiroinositol

It has been established that insulin treatment of cells, isolated plasma membranes, or whole animals leads to the generation of low molecular weight mediators which serve as intermediates in the signalling pathway. At least two distinct classes of mediator have been described, based on differences in apparent molecular weight, isoelectric point and biological activity (Cheng, K., and Larner, J. (1985) Ann. Rev. Physiol. 45, 407-424). Recently, Saltiel's (Saltiel, A.R., and Cuatrecasas, P. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5793-5797) and Mato's (Mato, J.M., Kelly, K.L., Abler, A., and Jarett, L. (1987) J. Biol. Chem. 262, 2131-2137) laboratories have described an insulin "modulator" which was apparently derived from glycosylphosphoinositol linker, similar to those known to anchor proteins to the external surface of the cell membrane (Low, M.G. (1987) Bioch. J. 244, 1-13). In this paper, we report that highly purified preparations of the insulin mediator which stimulates pyruvate dehydrogenase phosphatase contain mannose, galactosamine, and D-chiroinositol. These determinations are based upon analyses using paper chromatography and gas chromatography/mass spectroscopy. Nitrous acid deamination of the mediator resulted in release of inositol phosphate, indicating that the galactosamine and D-chiroinositol are linked. Although the presence of chiroinositol in modulator from H35 hepatoma cells has been recently reported (Mato, J.M., Kelly, K.L., Abler, A., Jarett, L., Corkey, B.E., Cashel, J.A., and Zopf, D. (1987) Bioch. Biophys. Res. Comm. 146, 764-770), the optical identity of the inositol remained unknown until the present report. Likewise, the presence of galactosamine rather than glucosamine in insulin mediator is a novel finding. These findings, coupled with those of Saltiel and Mato's groups, provide clear evidence for the existence of multiple forms of insulin mediators. Additionally, the results presented here afford further confirmation for the formation of insulin mediators from glycosyl-phosphoinositol linkers.

Analytical procedures for inflammation markers

The severity of inflammatory processes and the therapeutical approach today are mainly judged from the clinical status of a patient. Supplementary quantitative clinical and laboratory data mostly are related to the secondary reaction of the organism following the inflammatory stimulus. Typical measures are body temperature, white blood cell count, differential leukocytic hemogram, quantitative analysis of serum proteins, and quantification of acute phase proteins. The erythrocyte sedimentation rate also is a consequence of the altered serum protein pattern. The diagnostic usefulness of these parameters is limited for several reasons. The secondary reactions of the organism follow inflammatory stimuli with some delay, the data obtained in many cases are too late for initiation of an early therapy. On the other hand, the data obtained in many cases do not correlate quantitatively with the clinical severity of inflammatory processes. Therefore, the development of methodologies for quantitative measurement of parameters directly related to the inflammatory process ("Inflammation Markers") is a great challenge for pathobiochemical research. In the initiation, course, and termination of inflammatory processes humoral and cellular mechanisms are involved, which are interconnected to a complex network. Some important mechanisms involved are: Humoral mechanisms; coagulation and fibrinolysis; complement (classic and alternative pathway); kallikrein-kinin system. Cellular mechanisms; low molecular weight mediators; arachidonic acid metabolites; biogenic amines; oxygen metabolites. High molecular weight mediators; cytokins. Secretory proteins; lysosomal enzymes. In the preceding lectures the pathobiochemical significance of some of these mechanisms resp. products has been discussed. We present a review of the present and future repertoire of analytical procedures for parameters from these systems. The lecture will cover immunological, biochemical, chromatographic, as well as functional assays. Technical aspects and validity data regarding these assays are presented.

Studies on endothelial cell cytotoxic activity in sera of patients with progressive systemic sclerosis, Raynaud syndrome, rheumatoid arthritis, and systemic lupus erythematosus.

Using human umbilical cord endothelial cell cultures and a modified 3HTdR uptake technique, endothelial cell cytotoxic activity (ECA) has been demonstrated in sera of 95/130 patients with progressive systemic sclerosis (PSS), 14/20 patients with Raynaud syndrome (RS), 52/153 rheumatoid arthritis (RA), and 47/113 systemic lupus erythematosus (SLE) sera. ECA could be enriched by gel filtration from PSS sera in a molecular weight range of 5 k daltons. ECA was partially associated with serum proteins, mainly in the albumin containing fraction, albeit at a lower level of activity. In PSS, no relationship of ECA to the type of skin involvement was observed. ECA appears to be a low molecular weight mediator of, as yet, unknown origin.

Phagocytosis by mast cells in urticaria pigmentosa

Mast cells with endocytic and autophagic vacuoles were observed in a biopsy from a lesion of urticaria pigmentosa. In several areas of the specimen the mast cells revealed the typical morphology of mast cells which have released low molecular weight mediators, but this phenomenon did not seem to be correlated morphologically to the phagocytic activity of the mast cells. Some mast cells contained giant granules with a normal as well as an abnormal substructure.

Book Review

Book reviewed in this article:BASIC FOOD MICROBIOLOGY (1979). G. J. Banwart.BACTERIA AND HUMAN DISEASE (1979). J. M. Slack and I. S. Snyder.ANTIBIOTICS. Volume 5 (in two parts). PART 1: MECHANISM OF ACTION OF ANTIBACTERIAL AGENTS. PART 2: MECHANISM OF ACTION OF ANTIEUKARYOTIC AND ANTIVIRAL COMPOUNDS (1979). Part 1, 376 pp., Part 2, 470 pp. Edited by F. E. Hahn.COMBINED ANTIMICROBIAL THERAPY (1979). Edited by W. Brumfitt, L. Curcio and L. Silvestri.DEVELOPMENTAL BIOLOGY OF PROKARYOTES (1979). Edited by J. H. Parish.ADHESION OF MICRO‐ORGANISMS TO SURFACES (1979). Edited by D. C. Ellwood, J. Melling and P. Rutter.MICROBIAL CLASSIFICATION AND IDENTIFICATION (1980). Edited by M. Goodfellow and R. G. Board.FUNDAMENTALS OF FOOD CANNING TECHNOLOGY (1979). J. M. Jackson and B. M. Shinn.MICROTUBULES (1979). Edited by K. Roberts and J. S. Hyams.FOOD‐BORNE INFECTIONS AND INTOXICATIONS (1979). Edited by H. Riemann and F. L. Bryan.REGULATION OF MACROMOLECULAR SYNTHESIS BY LOW MOLECULAR WEIGHT MEDIATORS (1979). Edited by G. Koch and D. Richter.