Medical bioremediation of age-related diseases

Catabolic insufficiency in humans leads to the gradual accumulation of a number of pathogenic compounds associated with age-related diseases, including atherosclerosis, Alzheimer's disease, and macular degeneration. Removal of these compounds is a widely researched therapeutic option, but the use of antibodies and endogenous human enzymes has failed to produce effective treatments, and may pose risks to cellular homeostasis. Another alternative is "medical bioremediation," the use of microbial enzymes to augment missing catabolic functions. The microbial genetic diversity in most natural environments provides a resource that can be mined for enzymes capable of degrading just about any energy-rich organic compound. This review discusses targets for biodegradation, the identification of candidate microbial enzymes, and enzyme-delivery methods.     

Involvement of cis and trans Golgi apparatus elements in the intracellular sorting and targeting of acid hydrolases to lysosomes

To delineate the traffic route through the Golgi apparatus followed by newly synthesized lysosomal enzymes, we subfractionated the Golgi apparatus of rat liver by preparative free-flow electrophoresis into cisternae fractions of increasing content of trans face markers and decreasing contents of markers for the cis face. NADPase was used to mark median cisternae. Beta-Hexosaminidase, the high mannose oligosaccharide processing enzyme, alpha-mannosidase II, the two enzymes involved in the biosynthesis of the phosphomannosyl recognition marker, and the phosphomannosyl receptor itself decreased in specific activity or amount from cis to trans. Additionally, these activities were observed in a fraction consisting predominantly of cisternae, vesicles and tubules derived from trans-most Golgi apparatus elements. These results, along with preliminary pulse-labeling kinetic data for the phosphomannosyl receptor, suggest that lysosomal enzymes enter the Golgi apparatus at the cis face, are phosphorylated, and appear in trans face vesicles by a route whereby the phosphomannosyl receptor bypasses at least some median and/or trans Golgi apparatus cisternae.     

Evolution of arthropod hemocyanins and insect storage proteins (hexamerins)

Crustacean and cheliceratan hemocyanins (oxygen-transport proteins) and insect hexamerins (storage proteins) are homologous gene products, although the latter do not bind oxygen and do not possess the copper- binding histidines present in the hemocyanins. An alignment of 19 amino acid sequences of hemocyanin subunits and insect hexamerins was made, based on the conservation of elements of secondary structure observed in X-ray structures of two hemocyanin subunits. The alignment was analyzed using parsimony and neighbor-joining methods. Results provide strong indications for grouping together the sequences of the 2 crustacean hemocyanin subunits, the 5 cheliceratan hemocyanin subunits, and the 12 insect hexamerins. Within the insect clade, four methionine- rich proteins, four arylphorins, and two juvenile hormone-suppressible proteins from Lepidoptera, as well as two dipteran proteins, form four separate groups. In the absence of an outgroup sequence, it is not possible to present information about the ancestral state from which these proteins are derived. Although this family of proteins clearly consists of homologous gene products, there remain striking differences in gene organization and site of biosynthesis of the proteins within the cell. Because studies on 18S and 12S rRNA sequences indicate a rather close relationship between insects and crustaceans, we propose that hemocyanin is the ancestral arthropod protein and that insect hexamerins lost their copper-binding capability after divergence of the insects from the crustaceans.      

Interactions of Photobleaching and Inorganic Nutrients in Determining Bacterial Growth on Colored Dissolved Organic Carbon

Abstract Bacteria are key organisms in the processing of dissolved organic carbon (DOC) in aquatic ecosystems. Their growth depends on both organic substrates and inorganic nutrients. The importance of allochthonous DOC, usually highly colored, as bacterial substrate can be modified by photobleaching. In this study, we examined how colored DOC (CDOC) photobleaching, and phosphorus (P) and nitrogen (N) availability, affect bacterial growth. Five experiments were conducted, manipulating nutrients (P and N) and sunlight exposure. In almost every case, nutrient additions had a significant, positive effect on bacterial abundance, production, and growth efficiency. Sunlight exposure (CDOC photobleaching) had a significant, positive effect on bacterial abundance and growth efficiency. We also found a significant, positive interaction between these two factors. Thus, bacterial use of CDOC was accelerated under sunlight exposure and enhanced P and N concentrations. In addition, the accumulation of cells in sunlight treatments was dependent on nutrient availability. More photobleached substrate was converted into bacterial cells in P- and N-enriched treatments. These results suggest nutrient availability may affect the biologically-mediated fate (new biomass vs respiration) of CDOC.     

Retinoic acid and calcitriol inhibition of growth and NADH oxidase of normal and immortalized human keratinocytes.

Plasma membranes were isolated by aqueous two-phase partition from normal human keratinocytes (HKc) and from human keratinocytes immortalized with human papillomavirus type 16 DNA (HKc/HPV16). The NADH oxidase of plasma membrane vesicles of normal HKc was stimulated by epidermal growth factor whereas that of HKc/HPV16 was not. The NADH oxidase of the plasma membranes from both normal HKc and HKc/HPV16 was inhibited by calcitriol (1 alpha-1,25-dihydroxy vitamin D-3) and retinoic acid. However, with plasma membranes from HKc/HPV16 the NADH oxidase was more susceptible to inhibition by retinoic acid than were membranes from normal HKc. Similarly, clonal growth of HKc/HPV16 was inhibited by retinoic acid at lower concentrations than normal HKc whereas inhibition of clonal growth of normal HKc and HKc/HPV16 by calcitriol showed similar dose-dependencies. Comparing normal HKc and HKc/HPV16, the results demonstrate parallel inhibition of clonal growth and NADH oxidase by both retinoic acid and calcitriol of HKc/HPV16 but not of normal HKc. These results suggest that an increased sensitivity of the plasma membrane NADH oxidase of HKc/HPV16 to retinoic acid may be related to the increased sensitivity of these cells to growth control by retinoic acid. In addition, since plasma membrane NADH oxidase of HKc/HPV16 shows altered responsiveness to growth modulators such as EGF, retinoic acid and calcitriol, it appears that HKc/HPV16 express an NADH oxidase with different characteristics than those of normal HKc.