T tubules and surface membranes provide equally effective pathways of carbonic anhydrase-facilitated lactic acid transport in skeletal muscle

We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL) by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA) of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV-CAIX-CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli.     

Restricted aeroallergen access to airway mucosal dendritic cells in vivo limits allergen-specific CD4+ T cell proliferation during the induction of inhalation tolerance

Chronic innocuous aeroallergen exposure attenuates CD4(+) T cell-mediated airways hyperresponsiveness in mice; however, the mechanism(s) remain unclear. We examined the role of airway mucosal dendritic cell (AMDC) subsets in this process using a multi-OVA aerosol-induced tolerance model in sensitized BALB/c mice. Aeroallergen capture by both CD11b(lo) and CD11b(hi) AMDC and the delivery of OVA to airway draining lymph nodes by CD8α(-) migratory dendritic cells (DC) were decreased in vivo (but not in vitro) when compared with sensitized but nontolerant mice. This was functionally significant, because in vivo proliferation of OVA-specific CD4(+) T cells was suppressed in airway draining lymph nodes of tolerized mice and could be restored by intranasal transfer of OVA-pulsed and activated exogenous DC, indicating a deficiency in Ag presentation by endogenous DC arriving from the airway mucosa. Bone marrow-derived DC Ag-presenting function was suppressed in multi-OVA tolerized mice, and allergen availability to airway APC populations was limited after multi-OVA exposure, as indicated by reduced OVA and dextran uptake by airway interstitial macrophages, with diffusion rather than localization of OVA across the airway mucosal surface. These data indicate that inhalation tolerance limits aeroallergen capture by AMDC subsets through a mechanism of bone marrow suppression of DC precursor function coupled with reduced Ag availability in vivo at the airway mucosa, resulting in limited Ag delivery to lymph nodes and hypoproliferation of allergen-specific CD4(+) T cells.     

Distinctive regulation of the functional linkage between the human cation-independent mannose 6-phosphate receptor and GTP-binding proteins by insulin-like growth factor II and mannose 6-phosphate

The rat insulin-like growth factor II (IGF-II) receptor develops transmembrane signaling functions by directly coupling to a guanine nucleotide-binding protein (G protein) having a 40-kDa alpha subunit, Gi-2, whereas recent studies have indicated that the IGF-II receptor is a molecule identical to the cation-independent mannose 6-phosphate receptor (CI-MPR), a receptor implicated in lysosomal enzyme sorting. In this study, by using vesicles reconstituted with the clonal human CI-MPR and G proteins, we indicated that the CI-MPR could stimulate guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding and GTPase activities of Gi proteins in response to IGF-II. The stimulatory effect of IGF-II on Gi-2 depended on the reconstituted amount of the CI-MPR; it could not be found in vesicles reconstituted with Gi-2 alone; and it was also observed on Gi-1 reconstituted with the CI-MPR in phospholipid vesicles. Of interest, such stimulatory effect was not reproduced by Man-6-P in CI-MPR vesicles reconstituted with either G protein. Furthermore, the affinity for Man-6-P-mediated beta-glucuronidase binding to several kinds of native cell membranes was not reduced by 100 microM GTP gamma S. Instead, however, Man-6-P dose-dependently inhibited IGF-II-induced Gi-2 activation with an IC50 of 6 microM in vesicles reconstituted with the CI-MPR and Gi-2. The action of 100 nM IGF-II was completely abolished by 1 mM Man-6-P. Such an inhibitory effect of Man-6-P was reproduced by 4000 times lower concentrations of beta-glucuronidase or similar concentrations of fructose 1-phosphate, but not by mannose or glucose 6-phosphate. These results indicate that the human CI-MPR has two distinct signaling functions that positively or negatively regulate the activity of Gi-2 in response to the binding of IGF-II or Man-6-P.     

Carbonic anhydrase IV from human lung. Purification, characterization, and comparison with membrane carbonic anhydrase from human kidney

We have purified carbonic anhydrase (CA) IV from human lung membranes to apparent homogeneity in a form which is catalytically active and stable to storage. It has an apparent molecular mass of 35 kDa, is insensitive to endoglycosidases, and seems to contain no N-linked or O-linked oligosaccharide chains. Reduction of disulfide linkages led to altered migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and loss of catalytic activity. CA IV resembles CA II in being a "high activity" isozyme, relatively resistant to inhibition by halide ions and sensitive to inhibition by sulfonamides. Application of this purification to human kidney membranes produced homogeneous enzyme with nearly identical properties. Amino acid compositions of both lung and kidney CA IV were similar, as were tryptic peptide patterns resolved on high performance liquid chromatography (HPLC). Amino-terminal sequences of native enzyme from lung and kidney were identical, as were amino-terminal sequences of the three major tryptic peptides resolved on reverse phase HPLC. Isoelectric focusing revealed microheterogeneity in enzyme from both sources. Antibody raised to human lung CA IV reacted equally strongly with CA IV from kidney, but very weakly or not at all with other CAs. Treatment of lung membranes and kidney membranes with phosphatidylinositol-specific phospholipase C released over half of the membrane-bound CA IV, suggesting that at least half of the CA IV in both organs is anchored to membranes by phosphatidylinositol-glycan linkages.     

Binding of Colloidal MnO(2) by Extracellular Polysaccharides of Pedomicrobium manganicum

The extracellular acidic polysaccharides of the manganese-oxidizing bacterium Pedomicrobium manganicum were able to bind preformed colloidal MnO(2). The capacity of the cells to bind MnO(2) was pH dependent. Enhanced binding capacity below pH 5 suggests that ionic bonding forces are involved in the binding mechanism and that there is a charge reversal on the acidic polysaccharides between pH 5 and 4 that is due to increased protonation of carboxyl groups.     

Ecosystem health: an example of implications arising from regulations under the Canadian Environmental Protection Act (CEPA), and the importance of setting precedent

Ecosystem health is a key principle which underlies the Canadian Environmental Protection Act (CEPA). This act is designed to protect human health and the environment from harmful and/or irreversible effects by providing a cradle-to-grave regulation of toxic substances.As an example of the application of this act, this contribution considers the most toxic dioxins and furans which are generally associated with Kraft pulp mill effluents. These are some of the first substances to come under the CEPA legislation. The proposed CEPA regulations for dioxins and furans are based on end-of-pipe control, which would effectively limit their concentrations in effluent to something close to measurable levels. Depending on sample matrix and methodology, however, measurable levels may differ considerably.Evidence presented at the Alberta-Pacific (ALPAC) pulp mill hearings in Alberta and the Northwest Territories during 1989 demonstrated that total loading are particularly important in dealing with the far-field effects of these extremely toxic, persistent, and bioaccumulating substances. Further, it was reported that there might be no threshold of effect for tetrachlorodibenzo-para-dioxin (2, 3, 7, 8, TCDD) or the companion furan. If such evidence is correct, the CEPA regulations should be designed to achieve zero discharge of these contaminants. Measurable levels, as presently defined in the CEPA regulations, may be in excess of zero discharge requirements.Clearly, such inconsistency may cause problems and should be addressed directly. Unfortunately, the first draft of the CEPA regulations represents a piecemeal approach. In this, it is unfair to industry, it is scientifically inadequate, and it may not be enforceable. The application of CEPA regulations for the pulp and paper industry will set a new precedent for Canada's approach to ecosystem health. It is therefore essential to base decisions on a good under-standing of the dynamics and effects of chemicals in ecosystems and to re-evaluate, carefully, the toxicities of key contaminants. Interim measures are likely appropriate.     

PCR-mediated detection of acidophilic, bioleaching-associated bacteria.

The detection of acidophilic microorganisms from mining environments by culture methods is time consuming and unreliable. Several PCR approaches were developed to amplify small-subunit rRNA sequences from the DNA of six bacterial phylotypes associated with acidic mining environments, permitting the detection of the target DNA at concentrations as low as 10 fg.