Antimicrobial actions of dual oxidases and lactoperoxidase

The NOX/DUOX family of NADPH oxidases are transmembrane proteins generating reactive oxygen species as their primary enzymatic products. NADPH oxidase (NOX) 1–5 and Dual oxidase (DUOX) 1 and 2 are members of this family. These enzymes have several biological functions including immune defense, hormone biosynthesis, fertilization, cell proliferation and differentiation, extracellular matrix formation and vascular regulation. They are found in a variety of tissues such as the airways, salivary glands, colon, thyroid gland and lymphoid organs. The discovery of NADPH oxidases has drastically transformed our view of the biology of reactive oxygen species and oxidative stress. Roles of several isoforms including DUOX1 and DUOX2 in host innate immune defense have been implicated and are still being uncovered. DUOX enzymes highly expressed in the respiratory and salivary gland epithelium have been proposed as the major sources of hydrogen peroxide supporting mucosal oxidative antimicrobial defenses. In this review, we shortly present data on DUOX discovery, structure and function, and provide a detailed, up-to-date summary of discoveries regarding antibacterial, antiviral, antifungal, and antiparasitic functions of DUOX enzymes. We also present all the literature describing the immune functions of lactoperoxidase, an enzyme working in partnership with DUOX to produce antimicrobial substances.     

Synthesis of oligopeptides with the sequence SXWS and their chemotactic effects on a ciliated protozoan Tetrahymena pyriformis.

In this paper, the solid phase synthesis and chemical characterization of members of an SXWS sub-library (SAWS, SDWS and SKWS) as well as the comparison of their chemotactic properties with those of SEWS, which exhibits a prominent effect at 10(-12) M on a ciliated protozoan, Tetrahymena pyriformis, are described. We found that the chemotaxis of cells induced with the SXWS peptides varied according to the nature of the amino acid residue (Ala, Asp, Lys) in position X. The chemotactic activity of SEWS was not surpassed by any of three new tetrapeptides, although SAWS was also chemoattractant. Interestingly, SDWS, with an acidic side chain at position X, could not elicit any chemotactic response. SKWS, however, showed mild but significant chemorepellent activity over a wide concentration range. Chemotactic selection studies showed that the two chemoattractant peptides (SAWS and SEWS) had an expressed ability to select high-responder offspring cell populations. Peptides with neutral (SDWS) or chemorepellent (SKWS) properties were not able to select such subpopulations from the mixed cultures of Tetrahymena, indicating that the chemotactic response elicited by SXWS peptides is ligand-specific. For ligand-binding experiments N-terminally labelled fluorescent derivatives of SXWS peptides were prepared. applying [4-[7-hydroxycoumaryl]]acetic acid (Hca-OH) or 4-ethoxymethylene-2-[1]-naphthyl-5(4H)-oxazolone (naOx-OEt) as markers. Hca-OH was introduced using an active ester technique as the last step of SPPS, or after cleavage in solution. The oxazolone naOx-OEt reacted with the amino group of the peptide by liberation of EtOH. The binding characteristics of fixed Tetrahymena cells with the naOx-labelled peptides showed good correlation between binding profiles and chemotactic responsiveness (SEWS > SAWS > SDWS - SKWS). A similar binding pattern was observed in the case of Hca-peptides (SEWS > SAWS > SDWS). Hca-SKWS, however, bound remarkably to the cell surface. The binding activity of the Hca-peptides was less pronounced than that of the naOx-peptides, indicating the importance of the fluorophores applied.     

Characterisation of a GroEL Single-Ring Mutant that Supports Growth of Escherichia coli and Has GroES-Dependent ATPase Activity

Binding and folding of substrate proteins by the molecular chaperone GroEL alternates between its two seven-membered rings in an ATP-regulated manner. The association of ATP and GroES to a polypeptide-bound ring of GroEL encapsulates the folding proteins in the central cavity of that ring (cis ring) and allows it to fold in a protected environment where the risk of aggregation is reduced. ATP hydrolysis in the cis ring changes the potentials within the system such that ATP binding to the opposite (trans) ring triggers the release of all ligands from the cis ring of GroEL through a complex network of allosteric communication between the rings. Inter-ring allosteric communication thus appears indispensable for the function of GroEL, and an engineered single-ring version (SR1) cannot substitute for GroEL in vivo. We describe here the isolation and characterisation of an active single-ring form of the GroEL protein (SR-A92T), which has an exceptionally low ATPase activity that is strongly stimulated by the addition of GroES. Dissection of the kinetic pathway of the ATP-induced structural changes in this active single ring can be explained by the fact that the mutation effectively blocks progression through the full allosteric pathway of the GroEL reaction cycle, thus trapping an early allosteric intermediate. Addition of GroES is able to overcome this block by binding this intermediate and pulling the allosteric pathway to completion via mass action, explaining how bacterial cells expressing this protein as their only chaperonin are viable.     

Rate of phenotypic assortment in Tetrahymena thermophila

During vegetative, asexual reproduction in heterozygous Tetrahymena thermophila, the macronucleus divides amitotically to produce clonal lineages that express either one or the other allele but not both. Because such phenotypic assortment has been described for every locus studied, its mechanism has important implications concerning the development and structure of the macronucleus. The primary tools to study assortment are Rf/ the rate at which subclones come to express a single allele stably, and the output ratio, the ratio of assortee classes. Because Rf is related to the number of assorting units, a constant Rf for all loci suggests that all genes are maintained at the same copy number. Output ratios reflect the input ratio of assorting units, with a 1:1 output ratio implying equal numbers of alleles at the end of macronuclear development. Because different outcomes would suggest a different macronuclear structure, it is crucial that these parameters be accurately measured. Although published R_f values are similar for all loci measured, there has been no commonly accepted form of presentation and analysis. Here we examine the experimental determination of R_f. First, we use computer simulation to describe how the variability inherent in the assortment process affects experimental determination of Rf. Second, we describe a simple method of plotting assortment data that permits the uniform calculation of Rf, and we describe how to measure Rf accurately in instances when it is possible to score only the recessive allele. Using this method to produce truly comparable Rfs for all published data, we find that most, if not all, loci assort at Rfs consistent with ～45 assorting units, as has been asserted. © 1992 Wiley-Liss, Inc.     

A histochemical study of the muscles of Drosophila melanogaster.

The thoracic muscles of Drosophila melanogaster can be classified into two classes, the fibrillar and the tubular muscles, on morphological grounds. Histochemical techniques were used to characterize these two classes of muscle according to their content of various enzymes (alpha-glycerophosphate, NAD-dependent isocitrate, malate and succinate dehydrogenases, fumarase, acid phosphatase, adenosine triphosphatase and acetylcholinesterase) and of glycogen. These investigations showed that the two muslces types are histochemically very different and, further, that the morphologically similar tubular muscles are heterogeneous with respect to their enzyme content. In particular, the tergal depressor of the trochanter of the second leg, the largest of the tubular muslces, has considerably less of all the enzymes studied, with the exception of acetylcholinesterase, than all the other tubular muscles examined. The histochemical techniqes were also used to follow the changes in enzyme levels that occur during development of the indirect flight muscle fibres. All the enzymes that are present in adult flight muslces showed an increase in staining intensity throughout muscle development. Some minor differences were observed in the time of appearance and rate of increase of intensity of the different enzymes.     

Intranasal Delivery of Human β-Amyloid Peptide in Rats: Effective Brain Targeting

(1) Intranasal administration is a non-invasive and effective way for the delivery of drugs to brain that circumvents the blood–brain barrier. The aims of the study were to test a nasal delivery system for human β-amyloid (Aβ) peptides, to measure the delivery of the peptides to brain regions, and to test their biological activity in rats. (2) Aβ_1-42, in the form of a mixture of oligomers, protofibrils, and fibrils was dissolved in a nasal formulation containing hydrophobic, hydrophylic, and mucoadhesive components. The peptide solution was administered intranasally to rats as a single dose or in repeated doses. (3) Nasally injected Aβ labeled with the blue fluorescent dye amino-methyl coumarinyl acetic acid (AMCA) could be detected by fluorescent microscopy in the olfactory bulb and frontal cortex. The concentration of the peptide was quantified by fluorescent spectroscopy, and a significant amount of AMCA-Aβ peptide could be detected in the olfactory bulb. Unlabeled Aβ also reached the olfactory bulb and frontal cortex of rats as evidenced by intense immunostaining. (4) In behavioral experiments, nasal Aβ treatment did not affect anxiety levels (open-field test) and short-term memory (Y-maze test), but significantly impaired long-term spatial memory in the Morris water maze. The treatments did not result in Aβ immunization. (5) The tested intranasal delivery system could successfully target a bioactive peptide into the central nervous system and provides a basis for developing a non-invasive and cost effective, new model to study amyloid-induced dysfunctions in the brain.     

Testing use of a potential cognitive enrichment device by an Indo-Pacific bottlenose dolphin (Tursiops aduncus)

Cognitive enrichment aims to provide animals with opportunities to use their cognitive skills and to promote behaviors associated with positive wellbeing. Cooperation in mammals has been recorded during various behavioral contexts such as hunting, mating, playing, and parental care. Coordinated activity, often with some level of problem-solving action included, is required during cooperation. To investigate dolphins’ ability for collaborative problem-solving, an enrichment device was introduced to two adult male Indo-Pacific bottlenose dolphins (Tursiops aduncus). The device contained fish and ice and was designed to be opened by simultaneously pulling on both ends. After repeated presentation, it became apparent that only one dolphin had active interest in the device. To facilitate opportunities for problem-solving by this individual, an alternative collaborator, a human partner, was provided. Still, both dolphins had access to the device throughout the entire experiment. After the first opening, the same dolphin was highly successful in collaborating with the human in both joined (93%) and delayed (100%) partner conditions. The device provided a novel opportunity for the dolphin to use his cognitive skills. Even though only one dolphin participated actively, both dolphins showed varying degrees of interest to the device throughout the study. Both dolphins spent an average of 48% and 16% of their time, respectively, with the device, which resulted in a significant decrease in their other two most frequently observed behaviors: swimming and poolside observation. As a novel cognitive challenge, the device may be considered as a type of cognitive enrichment.