Methacholine-induced bronchoconstriction in rats: effects of intravenous vs. aerosol delivery

Peták, Ferenc, Zoltán Hantos, ÁgnesAdamicza, Tibor Asztalos, and Peter D. Sly. Methacholine-inducedbronchoconstriction in rats: effects of intravenous vs. aerosoldelivery. J. Appl. Physiol. 82(5):1479-1487, 1997.To determine the predominant site of action ofmethacholine (MCh) on lung mechanics, two groups of open-chestSprague-Dawley rats were studied. Five rats were measured duringintravenous infusion of MCh (iv group), with doubling of concentrationsfrom 1 to 16 µg · kg¹ · min¹.Seven rats were measured after aerosol administration of MCh with dosesdoubled from 1 to 16 mg/ml (ae group). Pulmonary input impedance(ZL) between 0.5 and 21 Hz wasdetermined by using a wave-tube technique. A model containing airwayresistance (Raw) and inertance (Iaw) and parenchymal damping (G) andelastance (H) was fitted to theZL spectra. In the iv group, MChinduced dose-dependent increases in Raw [peak response 270 ± 9 (SE) % of the control level; P < 0.05] and in G (340 ± 150%;P < 0.05), with no increase inIaw (30 ± 59%) orH (111 ± 9%). In the ae group, thedose-dependent increases in Raw (191 ± 14%;P < 0.05) andG (385 ± 35%; P < 0.05) were associated with a significant increase in H (202 ± 8%; P < 0.05).Measurements with different resident gases [air vs. neon-oxygenmixture, as suggested (K. R. Lutchen, Z. Hantos, F. Peták,Á. Adamicza, and B. Suki. J. Appl.Physiol. 80: 1841-1849, 1996)] in thecontrol and constricted states in another group of rats suggested thatthe entire increase seen in G during the ivchallenge was due to ventilation inhomogeneity, whereas the aechallenge might also have involved real tissue contractions viaselective stimulation of the muscarinic receptors.

     

Patterns of tolerance to heavy metals among methane-utilizing bacteria

The susceptibility of obligate methane-utilizing bacteria, including 14 reference strains and 175 environmental isolates, to five readily available heavy metal pollutants was determined. The chloride salts of Hg(II), Cd(II), Cu(II), Cr(III) and Zn(II) were tested in a system free from organic matter. Methane-utilizers appeared to have relatively discrete metal tolerance patterns with resistances to all metals varying with isolation site. Methanotrophs proved to be quite sensitive to mercury and cadmium but relatively resistant to copper, chromium and zinc     

Cloning and characterization of the human beta-glucuronidase gene

We have isolated a cosmid clone that contains GUSB, the human gene encoding beta-glucuronidase. The 21-kb gene contains 12 exons ranging from 85 to 376 bp in length. Exon 6 corresponds to the 153-bp deletion in the shorter of two types of cDNAs reported earlier, supporting the hypothesis that this cDNA arose by alternate splicing leading to exon skipping. The insert contains 4.2 kb of sequence upstream from the first exon and 6 kb 3' of the last exon. The clone expresses human beta-glucuronidase in stably transformed rat XCtk- cells. Comparison of the human gene organization with that recently reported for the murine beta-glucuronidase gene revealed that the intron/exon boundaries are identical. In the splice junctions, the most highly conserved regions are those identified as consensus sequences, and these are at least as highly conserved as bases encoding the translated portion of the gene.     

Biosynthesis and turnover of the phosphomannosyl receptor in human fibroblasts.

The phosphomannosyl receptor mediates intracellular targeting of newly synthesized acid hydrolases to lysosomes, and is also expressed as a pinocytosis receptor on the cell surface of fibroblasts. We have purified the phosphomannosyl receptor from bovine liver and produced rabbit antibodies to the bovine receptor. The antibodies partially blocked pinocytosis of human spleen beta-glucuronidase by fibroblasts, a process mediated by the phosphomannosyl receptor. Affinity-purified antibodies to the phosphomannosyl receptor were used to study the biosynthesis and turnover of the receptor in human fibroblasts. Phosphomannosyl receptor immunoprecipitated after a 15 min pulse-labelling of fibroblasts with [35S]methionine exhibited an identical mobility on sodium dodecyl sulphate/polyacrylamide gels as purified bovine liver phosphomannosyl receptor. Pulse-chase experiments for up to 3 days provided no evidence for changes in molecular weight attributable to post-translational processing of the phosphomannosyl receptor. Turnover studies determined that the half-life of the phosphomannosyl receptor in normal human fibroblasts was 24-29 h. The half-life of the receptor was slightly longer (32 h) in I-cell disease fibroblasts and normal fibroblasts exposed to leupeptin (32 h), slightly shorter in fibroblasts exposed to NH4Cl (23 h) and saturating amounts of ligand (21 h) and unaffected in cells exposed to mannose 6-phosphate (24 h). These studies show that the turnover of the phosphomannosyl receptor in fibroblasts is very slow, in contrast with its rate of internalization in endocytosis, and that its rate of degradation is not greatly altered by a variety of agents that affect lysosomal protein turnover and/or receptor-mediated endocytosis. These results suggest that the degradative activities of the lysosomes do not play an important role in phosphomannosyl receptor turnover in cultured fibroblasts.     

Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes

beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Most (90%) of the phosphomannosyl-enzyme receptors were found in endoplasmic reticulum, Golgi apparatus, and lysosomes; 9.5% in the plasma membrane, and less than 1% in nuclei and mitochondria. Receptors were vesicle-enclosed in all fractions except plasma membrane. Receptors in the endoplasmic reticulum apparently were occupied by endogenous ligands, but most receptors in lysosomes and plasma membrane were unoccupied. Most of the endogenous beta-hexosaminidase was in lysosomes and was released from vesicles by detergent treatment. Displacement of the residual receptor-bound endogenous beta-hexosaminidase (mostly in endoplasmic reticulum and Golgi apparatus) from detergent-treated membranes by mannose 6-phosphate released high uptake enzyme with properties expected for phosphomannosyl-enzymes. Mannose 6-phosphate-inhibitable enzyme receptor activity was found in nine rat organs and correlated roughly with their lysosomal enzyme content. These data support a general model for lysosomal enzyme transport in which the phosphomannosyl-enzyme receptor acts as a vehicle for delivery of newly synthesized acid hydrolases from the endoplasmic reticulum to lysosomes.     

Two unusual budding bacteria isolated from a swimming pool

Two unusual strains of budding bacteria were isolated on a Millipore Pseudomonas Count Water Tester during routine monitoring of Pseudomonas aeruginosa counts in a swimming pool. The first isolate has been identified as Blastobacter sp. It was a yellow-pigmented, Gram negative rod-shaped organism with a polar holdfast by which it attached to solid surfaces or other cells to form rosettes. The cells reproduced by asymmetric division or budding at the free pole of the cell, producing motile daughter cells with a single polar flagellum. The second isolate, which has not yet been identified, was a red-pigmented, Gram negative rod-shaped organism which produced one or more buds at each pole of the cell. Cell division appears to occur by both binary fission and by budding. Both organisms were strict aerobes, catalase and oxidase positive and did not produce acid from glucose in Hugh and Leifson medium.     

Frequency-dependent effects of hypercapnia on respiratory mechanics of cats

The effect of increasing arterial partial pressure of CO2 (PaCO2) on respiratory mechanics was investigated in six anesthetized, paralyzed cats ventilated by constant-flow inflation. Respiratory mechanics were studied after end-inspiratory occlusions. Zero frequency resistance (Rmax), infinite frequency resistance (Rmin), and static elastance (Est) were calculated for the respiratory system, lung, and chest wall. Alveolar ventilation was manipulated by the addition of dead space to achieve a range of PaCO2 values of 29.3-87.3 mmHg. Cats did not become hypoxic during the experiment. Under control conditions marked frequency dependence in Rmax, Rmin, and Est of the respiratory system, lungs, and chest wall was demonstrated. The chest wall contributed 50% of the total resistance of the respiratory system. With increasing PaCO2 the only resistance observed to increase was Rmax of the lung (P less than 0.01). There were also no changes in the static elastic properties of either the lungs or the chest wall. These results suggest that hypercapnia increases resistance by changes in the lung periphery and not in the conducting airways.     

Antibody to the phosphomannosyl receptor inhibits recycling of receptor in fibroblasts

The 215-kd phosphomannosyl receptor is involved in the transport of newly synthesized acid hydrolases to lysosomes and also mediates the pinocytosis of lysosomal enzymes by fibroblasts in culture. Recycling of receptors to the sorting sites is an integral part of both these processes. In this report, we describe the inhibition in human fibroblasts of both functions of the phosphomannosyl receptor by a rabbit antiserum to the bovine liver receptor. This inhibition cannot be completely accounted for by inhibition of ligand-receptor interaction. Rather the antibody appears to cross-link receptors and cause a removal of receptors from the sorting sites (plasma membrane and Golgi apparatus) and their accumulation in a compartment from which they do not recycle. Removal of receptors from the recycling pool by antibody is irreversible, and return of receptors requires synthesis of new protein. Degradation of "trapped receptors" is enhanced (t1/2 = 7.5 hr), but much more gradual than their removal from the functional receptor pool (t1/2 = 30 min).     

Sediment dispersion: part 2, characterisation by size of sand fraction and percent mud

In part 2 of this contribution, examples are drawn from the River Mersey and Liverpool Bay illustrating the use of simple statistical parameters to describe dispersion of sands and muddy sediments. The River Mersey and Liverpool Bay, eastern Irish Sea, were sites of intensive studies on the dispersal of dumped harbour mud and sewage sludge during the mid 1960's–70's. The combined effects of strong tidal scour, wave action and shoreward near-bed residual drift result in shoreward transport of large volumes of sand in the bay. Large amounts of mud (silt/clay mixtures) oscillate in the river estuary, and naturally derived and dumped muds also move shoreward in the bay. Unpublished historic geochemical data have been combined with reprocessed particle size data and both have been used to reassess sedimentological techniques for defining transport and dispersal pathways. River and bay muds have similar size compositions, but river muds have excess Cd > V > U > As = Zn relative to bay muds. The lower relative concentrations of heavy metals in the bay are thought to reflect desorption and degradation of organic matter from the river. Trends in sediment distribution data based on the means of the sand size fraction, alone, provide sensitivities comparable to those of higher order moment measures and are usually easier to interpret than full size spectrum analyses.