Effect of inhaled indomethacin on distilled water-induced airway epithelial cell swelling.

We evaluated the mechanism of the anti-asthmatic effect of inhaled indomethacin (Indo) by using an animal model (guinea pigs) of airway inflammation. After being exposed to either ozone or room air at identical flow rates (5 l/min) for 2 h, guinea pigs were anesthetized, tracheostomized, and lung resistance (RL) was subsequently measured. Guinea pigs inhaled either saline or Indo (1.5 mg/ml) for 1 min before undergoing an ultrasonically nebulized distilled water (UNDW) inhalation test. RL increased significantly after 10 min of UNDW inhalation in the room air and ozone groups but more so in the ozone group. This increase in RL was significantly suppressed by pretreatment with Indo. In the morphometric assessment of airway mucosa, a significant swelling of the epithelial cells after UNDW inhalation was observed in both the room air and ozone groups but especially so in the ozone group. This increase was also suppressed with Indo pretreatment. These results suggest that the increase in RL and the swelling of airway epithelial cells induced by inhaled UNDW in ozone-exposed guinea pigs was suppressed by pretreatment of inhaled Indo and that this suppression may be one of the reasons for the anti-asthmatic effect of inhaled Indo.     

Colonization process of the root endophytic fungus Heteroconium chaetospira in roots of Chinese cabbage

Dark septate endophytic fungi (DSE) may have an important functional relationship with host plants, but these functions and the colonization process remain unknown. We made microscopic observations of the growth of an endophytic hyphomycete in Chinese cabbage roots to understand its colonization process. This hyphomycete was Heteroconium chaetospira, a suspected DSE. Three weeks post inoculation, some hyphae became irregularly lobed and formed microsclerotia within host epidermal cells of healthy plants. In stunted plants, hyphae formed closely packed masses of fungal cells within host epidermal cells, but conidiophores rarely broke through the cell walls to produce conidia. Received: December 7, 2000 / Accepted: November 20, 2001     

Structure of human holocarboxylase synthetase gene and mutation spectrum of holocarboxylase synthetase deficiency

Holocarboxylase synthetase (HLCS) is an enzyme that catalyzes the incorporation of biotin into apo-carboxylases, and its deficiency causes biotin-responsive multiple carboxylase deficiency. The reported sequences of cDNA for human HLCS from liver, lymphocyte, and KG-1 myeloid cell lines differ at their 5' regions. To elucidate variations of the human HLCS mRNA and longer 5' cDNA ends, we performed screening of the human liver cDNA library and rapid amplification of the cDNA ends (RACE). Our results suggest the existence of three types of HLCS mRNA that start at different exons. The first type starts at exon 1, and the second type starts at exon 3, and both are found in various human tissues. The third type, corresponding to the cDNA from the KG-1 cell, starts at exon 2 of the HLCS gene. Various splicing patterns from exons 3-6 were also observed. None of the variations of cDNA found created a new initiation codon. Mutation screening from exons 6-14, therefore, was sufficient to detect amino acid changes in HLCS in patients. Our direct sequencing strategy for screening mutations in the HLCS gene revealed mutations in five Japanese patients and seven non-Japanese patients. Our analyses involving 12 Japanese and 13 non-Japanese patients and studies by others indicate that (1) there is no panethnically prevalent mutation; (2) the Arg508Trp, Gly581Ser, and Val550Met mutations are found in both Japanese and non-Japanese populations; (3) the IVS10+5G-->A mutation is predominant and probably a founder mutation in European patients; (4) the 655-656insA, Leu237Pro, and 780delG mutations are unique in Japanese patients; (5) the spectrum of the mutations in the HLCS gene may vary substantially among different ethnic groups.     

Studies on Tetrahymena membranes:: Temperature-induced alterations in fatty acid composition of various membrane fractions in Tetrahymena pyriformis and its effect on membrane fluidity as inferred by spin-label study

The fatty acid distribution pattern of lipids extracted from different subcellular components of Tetrahymena pyriformis was found to be significantly different from one type of membrane to another.The growth-temperature shift caused alterations in fatty acid composition. The ratio of palmitoleic to palmitic acid, especially, showed a sharp linear decline with increase of temperature in all of the membrane fractions.The spin labels were rapidly incorporated into Tetrahymena membranes. The order parameter of 5-nitroxide stearate spin label incorporated into various membrane fractions was found to be different for the different membrane fractions, suggesting the following order of the fluidity; microsomes > pellicles > cilia.The fluidity of the surface membranes, cilia and pellicles isolated from Tetrahymena cells grown at 15°C was noticeably higher than that of the membranes from cells grown at 34°C but was not so different with microsomal fractions.The motion of the spin label in the pellicular membrane was more restricted than in its extracted lipids, thus indicating the assumption that in Tetrahymena membranes the proteins influence the fluidity.It was also suggested that a sterol-like triterpenoid compound, tetrahymanol, which is principally localized in the surface membranes, would be involved in the membrane fluidity.     

Surface potential of phosphatidylserine monolayers II. Divalent and monovalent ion binding

Ion binding constants for phosphatidylserine membranes have been derived from the variation of the surface potential of phosphatidylserine monolayers with divalent cation concentrations in the presence of various monovalent salts in the aqueous subphase. The observed surface potential data for the monolayers, analyzed by use of the Gouy-Chapman diffuse potential theory, together with a simple binding reaction formula, yield, for Ca²⁺, Mg²⁺, Na⁺ and (Me)₄N⁺ binding constant values of 30 M^?1, 10 M^?1, 0.6 M^?1 and 0.05 M^?1, respectively. The effect of pH on surface potential of phosphatidylserine monolayers was found to be dependent upon ionic species other than H⁺ in the subphase solution. The distinction between apparent and intrinsic dissociation constants of H⁺ for biomolecules was made in terms of ion binding due to other ions at the same site as for H⁺ in biomolecules.     

Hydrophobic interaction between the monomer of mitochondrial malate dehydrogenase and phospholipid membranes.

Porcine mitochondrial malate dehydrogenase (EC 1.1.1.37) dissociates into subunits on dilution. The enzyme monomer caused large increases in the surface pressure of monolayers of 1:1 phosphatidylserine/phosphatidylcholine at air/water and oil/water interfaces. The monomer increased the permeability of phospholipid vesicles to 22Na+. Both effects were significantly greater than the corresponding effects of ribonuclease A, cytochrome c and the dimeric form of malate dehydrogenase. Changes in the circular-dichroism spectra of the enzyme indicated that conformational changes may be associated with dimer formation or when monomer interacts with lysophosphatidyl-choline. Similar interactions to those described may occur in situ when mitochondrial malate dehydrogenase is transported to the mitochondrial matrix from its site of synthesis on cytosolic ribosomes.     

A 3-Mb Sequence-Ready Contig Map Encompassing the Multiple Disease Gene Cluster on Chromosome 11q13.1-q13.3

Despite the presence of several human disease genes on chromosome11q13, few of them have been molecularly cloned. Here, we reportthe construction of a contig map encompassing 11q13.1–q13.3using bacteriophage P1 (P1), bacterial artificial chromosome(BAC), and P1-derived artificial chromosome (PAC). The contigmap comprises 32 P1 clones, 27 BAC clones, 6 PAC clones, and1 YAC clone and spans a 3-Mb region from D11S480 to D11S913.The map encompasses all the candidate loci of Bardet-Biedlesyndrome type I (BBS1) and spinocerebellar ataxia type 5 (SCA5),one-third of the distal region for hereditary paraganglioma2 (PGL2), and one-third of the central region for insulin-dependentdiabetes mellitus 4 (IDDM4). In the process of map construction,61 new sequence-tagged site (STS) markers were developed fromthe Not I linking clones and the termini of clone inserts. Wehave also mapped 30 ESTs on this map. This contig map will facilitatethe isolation of polymorphic markers for a more re.ned analysisof the disease gene region and identi.cation of candidate genesby direct cDNA selection, as well as prediction of gene functionfrom sequence information of these bacterial clones.     

Involvement of the membrane fluidity of lactosylceramide-targeted liposomes in their intrahepatic uptake

Incorporation of N-lignoceroyldihydrolactocerebroside (lactosylceramide) enhanced liver uptake of small unilamellar liposomes consisting of dipalmitoylphosphatidylcholine, cholesterol and dicetyl phosphate (molar ratio, 4:5:1). The increase in liver uptake was mostly accounted for by an enhanced uptake into the parenchymal cells. The enhancing effects of lactosylceramide on uptake of the liposomes into liver in vivo and into isolated parenchymal cells in vitro were greater with dipalmitoylphosphatidylcholine-based liposomes than with dimyristoylphosphatidylcholine-based ones. In contrast, addition of lactosylceramide had no significant effect on egg phosphatidylcholine vesicle uptake. The stimulated uptake of lactosylceramide liposomes by parenchymal cells was counteracted by added asialofetuin. These observations suggest that transfer of the targeted liposomes via a galactose-specific receptor into parenchymal cells may be controlled by the membrane fluidity of the liposomes.