Phenol oxidase in the blood cells of millipedes

The nature and properties of the phenol oxidase present in the blood cells and plasma of three species of millipedes, Thyropygus poseidon, Polydesmus species, and Spirostreptus asthenus, have been investigated using a number of substrates as well as activators and inhibitors. The enzyme is located in the granular haemocytes. In the in situ condition it oxidizes diphenols, polyphenols, and also tyrosine. But when extracted from the homogenate of blood cells it showed only diphenolase activity. There is evidence of phenol oxidase activity in the plasma, but it did not act on tyrosine. The results obtained have been discussed in the light of previous work. It is suggested that the cell enzyme may have two sites of activity responsible for the oxidation of diphenol and also tyrosine. The observation that the monophenol oxidase activity is absent when the enzyme is extracted and isolated suggests that one of the sites of activity of the enzyme may be destroyed in the process of extraction.     

Phenol oxidation in soft cuticle and blood of crayfish compared with that in other arthropods and activation of the phenol oxidases by fungal and other cell walls

Phenol oxidase from blood as well as from soft cuticle of different crayfish was highly activated by cell walls of a number of fungi and other plants, but the enzymes from other crustaceans tested and from some insects were not. Attachment of the phenol oxidase on blood and soft cuticle to the cell wall surface of certain plants and subsequent heavy melanization of the walls with natural or added substrate may be a unique property of crayfish since it did not occur in any other arthropods tested. Interestingly, blood and cuticle of crayfish showed the same specificity in these processes and the mechanisms might therefore be similar, if not the same, in both. The activity of the nonadsorbed enzyme dispersed in the blood was higher in a crayfish species resistant to fungal attack than in susceptible ones, but the relative degree of activation by adding cell walls was greater in the latter.The natural substrate for the phenol-oxidase activity in crayfish blood was released or mobilized by the blood cells. The substrate for the activity in cuticle was not available in the intact cuticle itself. It appeared only after a “signal” had been transmitted through the cuticle to the cells inside. The possibility that these mechanisms are involved in the cuticular as well as the blood defense system against fungal and other microbial parasites is discussed.     

Phenol oxidase activity: inductionin female schistosomes by in vitro incubation.

A biochemical methods has been developed for detecting phenol oxidase in female Schistosoma mansoni. Enzyme activity is observed only after incubation of the female schistosomes for an extended period of time in tissue culture media. Male S. mansoni do not contain detectable levels of phenol oxidase activity. The properties of this enzyme are similar to those identified for a phenol oxidase from Fasciola hepatica. L-DOPA, dopamine, and tyrosine were found to be good substrates for this enzyme. Vmax = 14.1, 8.1, and 6.1 mumoles O2/min/mg protein for each substrate, respectively. This enzyme appears to be associated with egg production and thus may be a useful marker for biochemical and immunological studies.     

Cellular export of sugars and amino acids: role in feeding other cells and organisms

Sucrose, hexoses, and raffinose play key roles in the plant metabolism. Sucrose and raffinose, produced by photosynthesis, are translocated from leaves to flowers, developing seeds and roots. Translocation occurs in the sieve elements or sieve tubes of angiosperms. But how is sucrose loaded into and unloaded from the sieve elements? There seem to be two principal routes: one through plasmodesmata and one via the apoplasm. The best-studied transporters are the H⁺/SUCROSE TRANSPORTERs (SUTs) in the sieve element-companion cell complex. Sucrose is delivered to SUTs by SWEET sugar uniporters that release these key metabolites into the apoplasmic space. The H⁺/amino acid permeases and the UmamiT amino acid transporters are hypothesized to play analogous roles as the SUT-SWEET pair to transport amino acids. SWEETs and UmamiTs also act in many other important processes—for example, seed filling, nectar secretion, and pollen nutrition. We present information on cell type-specific enrichment of SWEET and UmamiT family members and propose several members to play redundant roles in the efflux of sucrose and amino acids across different cell types in the leaf. Pathogens hijack SWEETs and thus represent a major susceptibility of the plant. Here, we provide an update on the status of research on intercellular and long-distance translocation of key metabolites such as sucrose and amino acids, communication of the plants with the root microbiota via root exudates, discuss the existence of transporters for other important metabolites and provide potential perspectives that may direct future research activities.

An update on intercellular and long-distance translocation of sugars and amino acids, including plant-root microbiota communication, other metabolite transporters is provided, and perspectives are discussed.     

Activation of NADPH oxidase 1 in tumour colon epithelial cells

In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22(phox), NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium) or NADP(+). The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1(Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O(2)(-)-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N-Rac1(Q61L)] between truncated NOXA1(1-211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox1 activity, but NOXO1N(1-292) [C-terminal truncated NOXO1(1-292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox1 activation and their interactions might be responsible for regulating the O(2)(-)-producing activity in Caco-2 cells.     

Phenol oxidase activation in Drosophila: A cascade of reactions

Our earlier evidence concerning the complexity of the activation process for Drosophila phenol oxidase has been confirmed by separation and purification of six proteins involved. This is a minimal number required in a reaction series or cascade to yield active enzyme, and at least two more proteins are known to be involved. A simpler system involving only the last step with one precursor and one activation as has been reported in the literature is consistent with the cascade picture, but the whole complex system must be considered when dealing with genetic and developmental regulation of pigmentation and sclerotization. Details are given for partial or complete purification of six of the proteins involved and evidence for their modes of interaction is presented.     

Activation of polyphenol oxidase by polyamines.

Latent polyphenol oxidase was extracted and partially purified from grape cell suspension cultures. The enzyme was shown to be activated by polyamines. Activation of the enzyme increased with increasing polyamine concentrations and half-maximal activation was in the order of 8mM. Kinetic parameters, Km and Vm, were also calculated for the latent and activated enzymes. The activating effect of polyamines was studied at different pH values. Optimum pH was 4.5 for latent and activated enzymes. However, the highest degree of activation was obtained at pH 5. Activation caused a higher sensitivity of polyphenol oxidase to pH and temperature. The ability of polyamines to activate the enzyme may suggest a limited conformational change.     

Concanavalin A mediated attachment and ingestion of red blood cells by macrophages.

The interaction of red blood cells and macrophages mediated by Concanavalin A (ConA) was studied using mouse peritoneal macrophages and fresh, homologous red cells. Erythrocytes exposed to ConA at 0.5 μg/ml, a condition that leads to a saturation of 3% of the ConA sites, were bound by macrophages at 22 °C. The ConA inhibitor, α-methylmannoside, prevented this attachment of red cells and largely reversed it when added to preformed macrophage-red cell rosettes up to 90 min. However, red cell attachment was essentially irreversible by 3 h. Electron microscopy showed a progressive increase in the degree of contiguity between red cells and macrophages with time, some macrophage projections distorting and partially encircling red cells at 3 h. Macrophages pretreated with high concentrations of ConA (25 μg/ml) also bound red cells. However, phagocytosis of adherent red cells did not occur at either 22 or 37 °C, even when both red cells and macrophages were pretreated with ConA. In contrast, phagocytosis of attached red cells was observed when preformed rosettes were exposed to ConA at a concentration of 5 μg/ml, and it was complete with ConA at a concentration of 25 μg/ml. These studies demonstrate that ConA in low concentration on red cells is detected by macrophages which form a progressively tighter bond with the red cell surface. However, it appears that phagocytosis can occur only under conditions in which a high density of ConA is established on the surface of the red cell.     

Encapsulation of foreign particles in vitro by separated blood cells from crayfish,Astacus leptodactylus

Summary Bone matrix consists of type-I collagen and noncollagenous proteins. The latter represent only 10% of its total protein content. Since type-I collagen is also present in various other connective tissue sites (e.g., skin) it cannot be considered as bone specific. Among the non-collagenous components osteonectin — a 32 kilodalton (KD) glycoprotein linking mineral to collagen fibrils — is thought to be bone specific due to its biochemical properties. In the present study various skeletal and non-skeletal tissues were investigated for the presence of osteonectin by means of immunocytochemical methods. Two polyclonal antibodies against human and bovine osteonectin were applied. Immunocytochemically, osteonectin could be demonstrated in active osteoblasts and osteoprogenitor cells as well as in young osteocytes, while aged, quiescent osteocytes did not contain the protein, suggesting that the protein is a marker of the osteoblastic functional differentiation of bone cells. Osteonectin was absent in all non-skeletal tissues with the exception of chondrocytes in so-called mineralizing chondroid bone.     

Disruption of bacterial quorum sensing by other organisms

Higher plants and algae produce compounds that mimic quorum sensing: signals used by bacteria to regulate the expression of many genes and behaviors. Similarly, various bacteria can stimulate, inhibit or inactivate quorum sensing in other bacteria. These discoveries offer new opportunities to manipulate bacterial quorum sensing in applications relevant to medicine, agriculture and the environment.     

Activation of cyclic AMP-dependent protein kinase by VIP in blood mononuclear cells

Vasoactive intestinal peptide stimulated cyclic AMP-dependent protein kinase activity in human blood mononuclear cells. The simultaneous presence of a phosphodiesterase inhibitor was required to elicit maximal activation. The apprent K_a value of half the maximal stimulation was about 60 pmol. Secretin exhibited a 170-times lower potency. Other peptides such as glucagon or insulin had no effect event at 1 μM.     

The regulation of superoxide production by the NADPH oxidase of neutrophils and other mammalian cells

Superoxide is produced by a NADPH oxidase of phagocytic cells and contributes to their microbicidal activities. The oxidase is activated when receptors in the neutrophil plasma membrane bind to the target microbe. These receptors recognise antibodies and complement fragments which coat the target cell. The oxidase electron transport chain, located in the plasma membrane, comprises a low potential cytochrome b heterodimer (gp 91-phox and p22-phox) associated with FAD. It is non-functional until at least three proteins, p67-phox, p47-phox and p21rac (and possibly others), move from the cytosol to dock on the cytochrome b. The docking involves the interaction of SH₃ domains may become exposed follwoing phosphorylation of p47-phox by protein kinase C or, in model systems, by addition of arachidonic acid to reconstitution mixtures. Following the docking process the electron-transporting component is able to transfer electrons from NADPH to oxygen. This electrogenic event is charge-compensated by the opening of a prton channel. Components of the oxidase are expressed in non-phagocytes, where their function is uncretain but could be related to some signal function of superoxide.     

Expression of components of the superoxide generating NADPH oxidase by human leucocytes and other cells

Summary NADPH oxidase of phagocytic leucocytes contains a membrane cytochromeb with two subunits, gp91 ^phox and p22 ^phox , together with three cytosolic proteins, p47 ^phox , p67 ^phox and p2 ^rac . The presence of some of these components has been sought in non-phagocytes, using Western blot analysis for protein expression and PCR to amplify and detect mRNA. All components were detected in EBV-transformed B lymphocytes and peripheral blood B lymphocytes. Fibroblasts and human kidney mesangial cells contained mRNA for p67 ^phox , p47 ^phox , and p22 ^phox but not gp91 ^phox . Levels of expression varied with growth conditions, but it appears possible than an isozyme of cytochromeb which lacks gp9 ^phox is present in these cells. Proteins of p47 ^phox and p67 ^phox were expressed, in low concentrations, in these two cell types. Expression of mRNA for p47 ^phox and p67 ^phox was found to be widespread in many cell types.Abbreviations IL-1 interleukin 1 - PMA phorbol myristate acetate - CGD chronic granulomatous disease - EBV-BL Epstein-Barr virus transformed B-lymphocytes - PBBL peripheral blood B lymphocytes