Biological degradation of explosives and chemical agents

Hazardous energetic organo-nitro compounds are found as contaminants in many environments. A series of nitro aromatics, nitrate esters and nitro amines, all characteristic of this class, has been studied for their susceptibility to biological transformation. Biotransformation pathways for each of these compounds have been identified and are summarized. Implications for these findings in light of current contamination issues is discussed. The detoxification of organophosphate chemical agents focuses on the investigation of organophosphate degrading enzymes from bacteria. Certain of these enzymes, active both in solution and when immobilized onto a solid surface, are very successful in hydrolyzing and detoxifying various organophosphate chemical agents. The relationship of this research to the critical concerns of the agricultural industry regarding detoxification of organophosphorous pesticides is discussed.     

Histochemical studies on the distribution of adenosine triphosphatase and 5'-nucleotidase amongst the constituents of the thoracic ganglia and the associated nerve of Periplaneta americana.

The present study deals with the distribution of adenosine triphosphatase and 5'-nucleotidase in the various constituents of thoracic ganglia and associated nerve of Periplaneta americana. The localization of both the enzymes in the thoracic ganglia is identical. The neural lamella is devoid of any activity for both the enzymes. The ganglion cells are intensely positive at their borders. The neuronal cell surface and/or glial cell processes which envelope the neurons show intense activity for these enzymes. Adenosine triphosphatase and 5'-nucleotidase are present around "giant fibres" and small axons. The activity appears to confine itself in the sheaths. The cytoplasm and the nuclei of the neurons are devoid of enzymatic activity, whereas the nucleoli are slightly active. The nerves are positive for both the enzymes. The role of these enzymes at different sites has also been discussed.     

Exploring the specific features of interfacial enzymology based on lipase studies

Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.     

Implication for functions of the ectopic adipocyte copper amine oxidase (AOC3) from purified enzyme and cell-based kinetic studies

AOC3 is highly expressed in adipocytes and smooth muscle cells, but its function in these cells is currently unknown. The in vivo substrate(s) of AOC3 is/are also unknown, but could provide an invaluable clue to the enzyme's function. Expression of untagged, soluble human AOC3 in insect cells provides a relatively simple means of obtaining pure enzyme. Characterization of enzyme indicates a 6% titer for the active site 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor and corrected k(cat) values as high as 7 s(-1). Substrate kinetic profiling shows that the enzyme accepts a variety of primary amines with different chemical features, including nonphysiological branched-chain and aliphatic amines, with measured k(cat)/K(m) values between 10(2) and 10(4) M(-1) s(-1). K(m)(O(2)) approximates the partial pressure of oxygen found in the interstitial space. Comparison of the properties of purified murine to human enzyme indicates k(cat)/K(m) values that are within 3 to 4-fold, with the exception of methylamine and aminoacetone that are ca. 10-fold more active with human AOC3. With drug development efforts investigating AOC3 as an anti-inflammatory target, these studies suggest that caution is called for when screening the efficacy of inhibitors designed against human enzymes in non-transgenic mouse models. Differentiated murine 3T3-L1 adipocytes show a uniform distribution of AOC3 on the cell surface and whole cell K(m) values that are reasonably close to values measured using purified enzymes. The latter studies support a relevance of the kinetic parameters measured with isolated AOC3 variants to adipocyte function. From our studies, a number of possible substrates with relatively high k(cat)/K(m) have been discovered, including dopamine and cysteamine, which may implicate a role for adipocyte AOC3 in insulin-signaling and fatty acid metabolism, respectively. Finally, the demonstrated AOC3 turnover of primary amines that are non-native to human tissue suggests possible roles for the adipocyte enzyme in subcutaneous bacterial infiltration and obesity.     

Cell Adhesion and Proliferation on Sulfonated and Non-Modified Chitosan Films

Three types of chitosan-based films have been prepared and evaluated: a non-modified chitosan film bearing cationizable aliphatic amines and two films made of N-sulfopropyl chitosan derivatives bearing both aliphatic amines and negative sulfonate groups at different ratios. Cell adhesion and proliferation on chitosan films of C2C12 pre-myoblastic cells and B16 cells as tumoral model have been tested. A differential cell behavior has been observed on chitosan films due to their different surface modification. B16 cells have shown lower vinculin expression when cultured on sulfonated chitosan films. This study shows how the interaction among cells and material surface can be modulated by physicochemical characteristics of the biomaterial surface, altering tumoral cell adhesion and proliferation processes.     

Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases

Serine proteinases of human polymorphonuclear neutrophils play an important role in neutrophil-mediated proteolytic events; however, the non-oxidative mechanisms by which the cells can degrade extracellular matrix in the presence of proteinase inhibitors have not been elucidated. Herein, we provide the first report that human neutrophils express persistently active cell surface-bound human leukocyte elastase and cathepsin G on their cell surface. Unstimulated neutrophils have minimal cell surface expression of these enzymes; however, phorbol ester induces a 30-fold increase. While exposure of neutrophils to chemoattractants (fMLP and C5a) stimulates modest (two- to threefold) increases in cell surface expression of serine proteinases, priming with concentrations of lipopolysaccharide as low as 100 fg/ml leads to striking (up to 10-fold) increase in chemoattractant-induced cell surface expression, even in the presence of serum proteins. LPS-primed and fMLP-stimulated neutrophils have approximately 100 ng of cell surface human leukocyte elastase activity per 10(6) cells. Cell surface- bound human leukocyte elastase is catalytically active, yet is remarkably resistant to inhibition by naturally occurring proteinase inhibitors. These data indicate that binding of serine proteinases to the cell surface focuses and preserves their catalytic activity, even in the presence of proteinase inhibitors. Upregulated expression of persistently active cell surface-bound serine proteinases on activated neutrophils provides a novel mechanism to facilitate their egress from the vasculature, penetration of tissue barriers, and recruitment into sites of inflammation. Dysregulation of the cell surface expression of these enzymes has the potential to cause tissue destruction during inflammation.     

Oxidation of Polyamines and Brain Injury

Several amine oxidases are involved in the metabolism of the natural polyamines putrescine, spermidine, and spermine, and play a role in the regulation of intracellular concentrations, and the elimination of these amines. Since the products of the amine oxidase-catalyzed reactions - hydrogen peroxide and aminoaldehydes - are cytotoxic, oxidative degradations of the polyamines have been considered as a cause of apoptotic cell death, among other things in brain injury. Since a generally accepted, unambiguous nomenclature for amine oxidases is missing, considerable confusion exists with regard to the polyamine oxidizing enzymes. Consequently the role of the different amine oxidases in physiological and pathological processes is frequently misunderstood. In the present overview the reactions, which are catalyzed by the different polyamine-oxidizing enzymes are summarized, and their potential role in brain damage is discussed.     

Amine N-methyltransferases from rabbit liver

N-Methylation of amines has been ascribed to enzymes listed as amine N-methyltransferase, indolethylamine N-methyltransferase, and arylamine N-methyltransferase. All of these activities are accomplished by each of two related enzymes present in rabbit liver. The two N-methyltransferases have a very broad and overlapping specificity for primary and secondary amines. Both have a molecular mass of 30,000 daltons and react with an antibody formed to one of them, but have different isoelectric points.     

Expression of Functional Human Monoamine Oxidase A and B cDNAs in Mammalian Cells

Monoamine oxidase (MAO) A and B are important enzymes that metabolize biogenic amines throughout the body. Previous studies had suggested that both MAO A and B consist of two subunits of molecular masses of 63 and 60 kilodaltons, respectively. The cDNAs encoding one subunit of human liver MAO A and B have been expressed in mammalian cells by transfection of the individual clones. The proteins expressed from these cDNAs are shown to be catalytically active. Similar to the endogenous enzymes, the expressed MAO A prefers serotonin as a substrate and is sensitive to the inhibitor clorgyline. In contrast, the expressed MAO B prefers phenylethylamine as a substrate and is sensitive to the inhibitor deprenyl. These results suggest that a single polypeptide of MAO A (or B), existing as either a monomer or homodimer, is enzymatically active. The ability to obtain functional MAO A and B from their respective cDNA clones allows us to study further the structure and function relationships of these important enzymes.     

Amine oxidases in apoptosis and cancer

Amine oxidases, the major enzymes of biogenic amines metabolism, are considered to be biological regulators, especially for cell growth and differentiation. A primary involvement of amine oxidases in cancer growth inhibition and progression, especially by means of aldehydes, H(2)O(2) and other reactive oxygen species, the amine oxidase-mediated products of biogenic amines oxidation, has been demonstrated. Amine oxidases are involved in cancer growth inhibition because of the higher content in tumour cells of biogenic amines in comparison to normal cells. The cytotoxic effect can be explained by a damage to cell membranes and/or nuclei or, indirectly, through modulation of membrane permeability transition and therefore apoptosis. The oxidation products of biogenic amines appears to be also carcinogenic, while acrolein, produced from the oxidation of spermine and spermidine, should be a key compound both carcinogenic and cytotoxic. The cancer inhibition/promotion effect of amine oxidases could be explained by taking into consideration the full pattern of the enzyme content of the cell. The balance of amine oxidases and antioxidant enzymes appear to be a crucial point for cancer inhibition or progression. A long lasting imbalance of these enzymes appears to be carcinogenic, while, for a short time, amine oxidases are cytotoxic for cancer cells.     

Resuscitation-Promoting Factors Are Cell Wall-Lytic Enzymes with Important Roles in the Germination and Growth of Streptomyces coelicolor

Dormancy is a common strategy adopted by bacterial cells as a means of surviving adverse environmental conditions. For Streptomyces bacteria, this involves developing chains of dormant exospores that extend away from the colony surface. Both spore formation and subsequent spore germination are tightly controlled processes, and while significant progress has been made in understanding the underlying regulatory and enzymatic bases for these, there are still significant gaps in our understanding. One class of proteins with a potential role in spore-associated processes are the so-called resuscitation-promoting factors, or Rpfs, which in other actinobacteria are needed to restore active growth to dormant cell populations. The model species Streptomyces coelicolor encodes five Rpf proteins (RpfA to RfpE), and here we show that these proteins have overlapping functions during growth. Collectively, the S. coelicolor Rpfs promote spore germination and are critical for growth under nutrient-limiting conditions. Previous studies have revealed structural similarities between the Rpf domain and lysozyme, and our in vitro biochemical assays revealed various levels of peptidoglycan cleavage capabilities for each of these five Streptomyces enzymes. Peptidoglycan remodeling by enzymes such as these must be stringently governed so as to retain the structural integrity of the cell wall. Our results suggest that one of the Rpfs, RpfB, is subject to a unique mode of enzymatic autoregulation, mediated by a domain of previously unknown function (DUF348) located within the N terminus of the protein; removal of this domain led to significantly enhanced peptidoglycan cleavage.     

Autodisplay of functional CYP106A2 in Escherichia coli

Cytochrome P450 enzymes catalyse a wide variety of reactions, including the hydroxylation and epoxidation of CC bonds, and dealkylation reactions. There is high interest in these reactions for biotechnology and pharmaceutical processes. Many P450s require membrane surroundings and have substrates that do not cross biological membranes. To circumvent these obstacles, CYP106A2 from Bacillus megaterium was expressed on the outer membrane of Escherichia coli cells by Autodisplay. Exposure on the surface was confirmed by a protease accessibility test and flow cytometry after immunolabelling. HPLC assays showed that 0.5 ml of cells displaying the enzyme (OD??? = 6) converted 9.13 μmol of deoxycorticosterone to 15β-OH-deoxycorticosterone within 1h. Imipramine and abietic acid were also accepted as substrates. The number of active enzyme molecules per cell was calculated to be 20,000. Surprisingly, surface-exposed CYP106A2 was active in E. coli BL21 without the external addition of the heme group. However, when CYP106A2 was expressed on the surface of an E. coli strain lacking the TolC channel protein (JW5503), enzymatic activity was almost completely abolished. The activity of CYP106A2 on the surface of E. coli JW5503 could be restored by the external addition of the heme group. This suggests, as has been reported before, that E. coli uses a TolC-dependent mechanism to export heme into the growth media, where it can be scavenged by a surface-displayed apoenzyme. Our results indicate that Autodisplay enables the functional surface display of P450 enzymes and provides a new platform to access their synthetic potential.     

Effects of protease treatment on growth,morphology, adhesion,and cell surface proteins of secondary chick embryo fibroblasts

Several proteolytic enzymes have been studied with regard to their ability to induce DNA synthesis and cell proliferation in resting chick embryo fibroblasts. Of the enzymes examined, thrombin, bromelin, and trypsin exhibit potent mitogenic activity, elastase has significant but less marked activity, whereas thermolysin, papain, and α-protease are inactive. The enzymes were also tested for their ability to induce morphological change or to remove two iodinatable proteins of 250,000 and 205,000 daltons. Although the larger protein is removed by some but not all of the proteases examined, every protease tested removed the smaller cell surface protein. The ability of proteases to stimulate cell growth could not be correlated directly with removal of either of these cell surface proteins; however, loss of the smaller protein does correlate with the reduction of both cytoplasmic spreading and cell-cell interactions observed after protease treatment. A secondary, later event of migration of cells into clumps is observed in those instances when protease treatment did not result in a loss of the 250K protein. A role for each of these proteins in the processes of cellular adhesion is discussed.     

Evidence for anterograde transport of secretory granules in processes of gastric paracrine (somatostatin) cells

Certain disseminated endocrine-like cells have previously been found to give off long cytoplasmic processes which end with small bulbous expansions on the membranes of other cell types. It is believed that the process-carrying cells control the functions of the receiving cells by local and directed (paracrine) secretion of messenger molecules (peptides, biogenic monoamines) through their processes. Following injections of amine precursors paracrine cells take up and convert these to the corresponding amines, which can be cytochemically visualized by the Falck-Hillarp formaldehyde-induced fluorescence technique. As the amines are stored in the cytoplasmic (secretory) granules of the cells, they form useful markers for studies of granule turnover and transport. By injecting, at different time intervals, two different precursors (L-5-hydroxytryptophan and L-3,4-dihydroxyphenylalanine), resulting in amines giving different fluorescence colours in the Falck-Hillarp procedure, we have been able to separately label old and new secretory granule fractions in different fluorescence colours. Examination of such double-labelled paracrine cells (mostly gastric somatostatin cells) indicates that their secretory granules are transported in a proximo-distal direction in the paracrine cell processes (" paraxons "). This finding strongly supports the concept that paracrine cells control the functions of the cells they contact by way of directed, process-mediated delivery of their secretory products.     

Analysis of polyamines as markers of (patho)physiological conditions

The aliphatic polyamines, putrescine, spermidine and spermine, are normal cell constituents that play important roles in cell proliferation and differentiation. The equilibrium between cellular uptake and release and the balanced activities of biosynthetic and catabolic enzymes of polyamines are essential for normal homeostasis in the proliferation and functions of cells and tissues. However, the intracellular polyamine content increases in hyperplastic or neoplastic growth. Although the involvement of polyamines in physiological and pathological cell proliferation and differentiation has been well established, the role they play is quite different in relation to cell systems and animal models and is dependent on inducer agents and stimuli. Also, the experimental procedures used to deplete polyamines have been shown to influence the cell responses. In this paper, the assay methods currently in use for polyamines are reviewed and compared with respect to sensitivity, reproducibility and applicability to routine analysis. The relevance of polyamine metabolism and the uptake/release process in many physiological and pathological processes is highlighted, and the cellular polyamine pathways are discussed in relation to the possible diagnostic and therapeutic significance of these mediators.     

Inhibition of enzymes of polyamine biosynthesis by substrate-like O-substituted hydroxylamines

The biogenic amines spermine, spermidine, and putrescine are essential factors of cell growth and differentiation. To inhibit pyridoxal-5"-phosphate dependent ornithine decarboxylase and pyruvate dependent S-adenosylmethionine decarboxylase, key enzymes of polyamine biosynthesis, a system of substrate-like O-substituted hydroxylamines is suggested. The best of these compounds were active at nanomolar concentrations. High potency and specificity of this type of inhibitors are discussed in terms of structural similarity of E–I and E–S complexes.     

Molecular characterization of the ebony gene from the American cockroach, Periplaneta americana

Biogenic amines are an important class of primary messengers in the central (CNS) and peripheral nervous systems and in peripheral organs. These substances regulate and modulate many physiological and behavioral processes. Various inactivation mechanisms for these substances exist to terminate biogenic amine-mediated signal transduction. In vertebrates, the enzymes monoamine oxidase and/or catechol-O-methyl-transferase are involved in these processes. In insects, however, in which both enzymes are low in abundance or absent, biogenic amines are inactivated mainly by N-acetylation or O-sulphation. In Drosophila, beta-alanyl conjugation mediated by the Ebony protein has recently been shown to be a novel and alternative pathway for biogenic amine inactivation. Here, we report the cloning of ebony cDNA (Peaebony) from a brain-specific cDNA library of the cockroach Periplaneta americana. The open reading frame encodes a protein of 860 amino acid residues (PeaEbony). The PeaEbony polypeptide shares homology to Ebony sequences from Anopheles gambiae, Apis mellifera, and Drosophila melanogaster. In addition, PeaEbony exhibits sequence similarity to a family of microbial non-ribosomal peptide synthetases. The mRNA encoding PeaEbony is highly expressed in the cockroach brain and to a lesser extent in the salivary glands. PeaEbony is, therefore, probably involved in the inactivation of various biogenic amines through beta-alanyl conjugation in the cockroach CNS. Since the salivary glands in Periplaneta are innervated by dopaminergic and serotonergic neurons, PeaEbony probably also biochemically modifies dopamine and serotonin in these acinar glands.     

Cold active microbial lipases: some hot issues and recent developments

Lipases are glycerol ester hydrolases that catalyze the hydrolysis of triglycerides to free fatty acids and glycerol. Lipases catalyze esterification, interesterification, acidolysis, alcoholysis and aminolysis in addition to the hydrolytic activity on triglycerides. The temperature stability of lipases has regarded as the most important characteristic for use in industry. Psychrophilic lipases have lately attracted attention because of their increasing use in the organic synthesis of chiral intermediates due to their low optimum temperature and high activity at very low temperatures, which are favorable properties for the production of relatively frail compounds. In addition, these enzymes have an advantage under low water conditions due to their inherent greater flexibility, wherein the activity of mesophilic and thermophilic enzymes are severely impaired by an excess of rigidity. Cold-adapted microorganisms are potential source of cold-active lipases and they have been isolated from cold regions and studied. Compared to other lipases, relatively smaller numbers of cold active bacterial lipases were well studied. Lipases isolated from different sources have a wide range of properties depending on their sources with respect to positional specificity, fatty acid specificity, thermostability, pH optimum, etc. Use of industrial enzymes allows the technologist to develop processes that closely approach the gentle, efficient processes in nature. Some of these processes using cold active lipase from C. antarctica have been patented by pharmaceutical, chemical and food industries. Cold active lipases cover a broad spectrum of biotechnological applications like additives in detergents, additives in food industries, environmental bioremediations, biotransformation, molecular biology applications and heterologous gene expression in psychrophilic hosts to prevent formation of inclusion bodies. Cold active enzymes from psychrotrophic microorganisms showing high catalytic activity at low temperatures can be highly expressed in such recombinant strains. Thus, cold active lipases are today the enzymes of choice for organic chemists, pharmacists, biophysicists, biochemical and process engineers, biotechnologists, microbiologists and biochemists.