The biogenesis protein PEX14 is an optimal marker for the identification and localization of peroxisomes in different cell types, tissues, and species in morphological studies

Catalase and ABCD3 are frequently used as markers for the localization of peroxisomes in morphological experiments. Their abundance, however, is highly dependent on metabolic demands, reducing the validity of analyses of peroxisomal abundance and distribution based solely on these proteins. We therefore attempted to find a protein which can be used as an optimal marker for peroxisomes in a variety of species, tissues, cell types and also experimental designs, independently of peroxisomal metabolism. We found that the biogenesis protein peroxin 14 (PEX14) is present in comparable amounts in the membranes of every peroxisome and is optimally suited for immunoblotting, immunohistochemistry, immunofluorescence, and immunoelectron microscopy. Using antibodies against PEX14, we could visualize peroxisomes with almost undetectable catalase content in various mammalian tissue sections (submandibular and adrenal gland, kidney, testis, ovary, brain, and pancreas from mouse, cat, baboon, and human) and cell cultures (primary cells and cell lines). Peroxisome labeling with catalase often showed a similar tissue distribution to the mitochondrial enzyme mitochondrial superoxide dismutase (both responsible for the degradation of reactive oxygen species), whereas ABCD3 exhibited a distinct labeling only in cells involved in lipid metabolism. We increased the sensitivity of our methods by using QuantumDots?, which have higher emission yields compared to classic fluorochromes and are unsusceptible to photobleaching, thereby allowing more exact quantification without artificial mistakes due to heterogeneity of individual peroxisomes. We conclude that PEX14 is indeed the best marker for labeling of peroxisomes in a variety of tissues and cell types in a consistent fashion for comparative morphometry.     

Role of bicarbonate as a pH buffer and electron sink in microbial dechlorination of chloroethenes

ABSTRACT: BACKGROUND: Buffering to achieve pH control is crucial for successful trichloroethene (TCE) anaerobic bioremediation. Bicarbonate (HCO3-) is the natural buffer in groundwater and the buffer of choice in the laboratory and at contaminated sites undergoing biological treatment with organohalide respiring microorganisms. However, HCO3- also serves as the electron acceptor for hydrogenotrophic methanogens and hydrogenotrophic homoacetogens, two microbial groups competing with organohalide respirers for hydrogen (H2). We studied the effect of HCO3- as a buffering agent and the effect of HCO3--consuming reactions in a range of concentrations (2.5-30 mM) with an initial pH of 7.5 in H2-fed TCE reductively dechlorinating communities containing Dehalococcoides, hydrogenotrophic methanogens, and hydrogenotrophic homoacetogens. RESULTS: Rate differences in TCE dechlorination were observed as a result of added varying HCO3- concentrations due to H2-fed electrons channeled towards methanogenesis and homoacetogenesis and pH increases (up to 8.7) from biological HCO3- consumption. Significantly faster dechlorination rates were noted at all HCO3- concentrations tested when the pH buffering was improved by providing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as an additional buffer. Electron balances and quantitative PCR revealed that methanogenesis was the main electron sink when the initial HCO3- concentrations were 2.5 and 5 mM, while homoacetogenesis was the dominant process and sink when 10 and 30 mM HCO3- were provided initially. CONCLUSIONS: Our study reveals that HCO3- is an important variable for bioremediation of chloroethenes as it has a prominent role as an electron acceptor for methanogenesis and homoacetogenesis. It also illustrates the changes in rates and extent of reductive dechlorination resulting from the combined effect of electron donor competition stimulated by HCO3- and the changes in pH exerted by methanogens and homoacetogens.