Secondary release of thromboxane A2 in aerosol leukotriene C4-induced bronchoconstriction in guinea pigs

Effect of a thromboxane synthetase inhibitor (OKY-046) on bronchoconstriction induced by aerosol leukotriene C4 and histamine was studied in anesthetized, artificially ventilated guinea pigs in order to examine whether secondary release of thromboxane A2 is produced by aerosol leukotriene C4 or not. 0.01–1.0μg/ml of leukotriene C4 and 12.5–400μg/ml of histamine inhaled from ultrasonic nebulizer developed for small animals caused dose-dependent increase of pressure at airway opening (Pao) which is considered to be an index representing bronchial response. Pretreatment of the animals with intravenous OKY-046 (100mg/kg) significantly reduced the airway responses produced by inhalation of 0.1, 0.33 and 1.0μg/ml of leukotriene C4, while the pretreatment did not affect the histamine dose-response curve. Based on these findings and previous reports (6, 7), it is suggested that aerosol leukotriene C4 activates arachidonate cyclooxygenase pathway including thromboxane A2 synthesis and the released cyclooxygenase products have bronchodilating effect as a whole     

Increase in nucleoside diphosphatase in rat brain striatum lesioned with kainic acid

The activity of ammoniagenesis from guanine nucleotides was found to increase significantly in rat brain after infusion of kainic acid into the striatum. Among the enzymes involved in degrading guanine nucleotides, nucleoside diphosphatase was markedly increased in the lesioned striatum. The enzyme activity began to increase 2 days after the infusion, and reached the maximum on the 13th day, the level being 4 times as high as that of the intact contralateral region. The increased activity was due to Type L enzyme, judging from its substrate specificity. Puromycin and cycloheximide inhibited this increase, indicating that the increased activity resulted from an increase in the net synthesis of the enzyme. These findings suggest that Type L NDPase might play some important roles in gliosis after neuronal lesion.     

Molecular cloning of mouse beta 2-glycoprotein I and mapping of the gene to chromosome 11.

beta 2-Glycoprotein I (beta 2 GPI), a plasma protein that binds to anionic phospholipids, is composed of five repeating units called a short consensus repeat (SCR), which is found mostly in the regulatory proteins of the complement system. Recently the human beta 2 GPI gene has been assigned to chromosome 17, not to chromosome 1 where most of the genes of the SCR-containing proteins are clustered. In this report, we have isolated a full-length cDNA clone of mouse beta 2 GPI and determined the chromosomal localization of the gene. The amino acid sequence deduced from the nucleotide sequence of mouse beta 2 GPI revealed 76.1% identity with that of human beta 2 GPI. A genetic mapping by in situ hybridization and linkage analysis using 50 backcross mice has shown that the mouse beta 2 GPI gene (designated B2gp1) is located on the terminal portion of the D region of chromosome 11, closely linked to Gfap, and is 18 cM distal to Acrb, extending a conserved linkage group between mouse chromosome 11 and human chromosome 17. On the basis of these results, the evolutionary relationships among the SCR-containing proteins are discussed.     

An interspecific linkage map of mouse chromosome 15 positioned with respect to the centromere

We have used an interspecific backcross between C57BL/6J and Mus spretus to derive a molecular genetic linkage map of chromosome 15 that includes 25 molecular markers and spans 93% of the estimated length of chromosome 15. Using a second interspecific backcross that was analyzed with a centromere-specific marker, we were also able to position our map with respect to the chromosome 15 centromere. This map provides molecular access to many discrete regions on chromosome 15, thus providing a framework for establishing relationships between cloned DNA markers and known mouse mutations and for identifying homologous genes in mice and humans that may be involved in disease.     

A molecular genetic linkage map of mouse chromosome 18 reveals extensive linkage conservation with human chromosomes 5 and 18

An interspecific backcross between C57BL/6J and Mus spretus was used to generate a molecular genetic linkage map of mouse chromosome 18 that includes 23 molecular markers and spans approximately 86% of the estimated length of the chromosome. The Apc, Camk2a, D18Fcr1, D18Fcr2, D18Leh1, D18Leh2, Dcc, Emb-rs3, Fgfa, Fim-2/Csfmr, Gnal, Grl-1, Grp, Hk-1rs1, Ii, Kns, Lmnb, Mbp, Mcc, Mtv-38, Palb, Pdgfrb, and Tpl-2 genes were mapped relative to each other in one interspecific backcross. A second interspecific backcross and a centromere-specific DNA satellite probe were used to determine the distance of the most proximal chromosome 18 marker to the centromere. The interspecific map extends the known regions of linkage homology between mouse chromosome 18 and human chromosomes 5 and 18 and identifies a new homology segment with human chromosome 10p. It also provides molecular access to many regions of mouse chromosome 18 for the first time.     

Analysis of 2-amino-N6-hydroxyadenine-induced mutagenesis in phage M13mp2

The mechanism of mutagenesis induced by 2-amino-N6-hydroxyadenine (AHA) and its deoxyriboside (AHAdR) was studied by determining the nucleotide sequences of phage M13mp2 mutant DNA samples. Mutations in the lac promoter-lacZ alpha region of the phage were induced by addition of this agent to culture media in which the phage was growing inside the host bacteria. The spectrum of spontaneous mutation was also investigated. The induced sequence changes were mostly base transitions (80% with AHA and 90% with AHAdR). A few single-base deletions and additions were detected, but they were ascribable to spontaneous mutations. These results are consistent with the incorporation type mechanism proposed by Janion (this issue). In the Ames Salmonella assay, both AHA and AHAdR showed strong mutagenicity in strain TA100 but no activity in TA98.     

The influence of experimentally produced oligohydramnios on lung growth and pulmonary surfactant content in fetal rabbits.

To study the effect of oligohydramnios on lung growth and biochemical lung development in fetal rabbits, amniotic fluid was drained through a tube inserted into the maternal peritoneal cavity on the 23 day of gestation. Littermate fetuses without an amniotic shunt were used as controls. The fetuses were delivered abdominally on the 28 day of gestation. In a total of 8 pregnant does, 17 fetuses underwent amniotic shunting and 22 fetuses were used as controls. The amniotic shunt produced a significant reduction in the amniotic fluid volume. There were no differences in the wet weights of the fetal body, liver or brain between the two groups. However, the amniotic shunt significantly decreased the wet weight of the fetal lung, fetal lung wet weight/body weight ratio, and protein concentration per lung as compared to the control fetuses. In the fetal liver and brain tissues, no changes were found in the concentrations of total phospholipids, phosphatidylcholine (PC) or disaturated phosphatidylcholine (DSPC, the main component of lung surfactant) per g of wet tissue and per mg of protein. However, the lungs of the fetuses with amniotic shunts contained significantly more PC and DSPC, and the L/S ratio was higher than in the control fetuses. These results suggest that the oligohydramnios produced by an amniotic shunt causes pulmonary hypoplasia, but raises the pulmonary surfactant content of fetal rabbit lung.     

The root-knot nematode resistance gene (Mi) in tomato: construction of a molecular linkage map and identification of dominant cDNA markers in resistant genotypes

A dominant allele at the Mi locus on chromosome 6 of tomato (Lycopersicon esculentum Mill) confers resistance to three species of root-knot nematodes (Meloidogyne). The resistance, which is associated with a localized necrotic response, was originally introduced into tomato from the wild species Lycopersicon peruvianum. As a step towards the molecular cloning of Mi, we have identified closely linked DNA markers from both cDNA and genomic DNA libraries as restriction fragment length polymorphisms (RFLPs). DNA from tomato populations segregating for nematode resistance was analyzed to generate a high-resolution genetic map of this region. Additional information on gene order was obtained by comparing the size of the introgressed L. peruvianum chromosomal segment within a collection of nematode-resistant tomato lines. Among the four cDNA markers that are tightly linked to Mi, three are dominant, i.e. L. peruvianum-specific. One cDNA marker corresponds to a gene family comprising 20-30 members, one of which is diagnostic for all nematode-resistant genotypes tested. The presence of non-homologous sequences around the Mi gene may contribute to the suppression of recombination in this region of the genome in crosses heterozygous for Mi. The potential of 'walking' from closely linked markers to Mi is discussed.     

Enhancement of tissue plasminogen activator production from human normal fibroblast IMR-90 cells by limitation of cell growth

Summary Tissue plasminogen activator (t-PA) production induced by proteose peptone from IMR-90 cells was investigated. Cells monolayered on plastic surfaces had a higher ability to produce t-PA per unit cell compared to those grown tri-dimensionally on ceramic pieces. Furthermore, confluent monolayers of the cells, which suffered contact inhibition and resulted in limited growth, were available for t-PA production. Repeated batch production with microcarriers, on which the cells were almost confluent monolayers similar to those in T-flasks, was performed. Utilization of the cells, which had limited serum in the growth phase, resulted in an increase in production. Moreover, dilution of the basal components of the medium at initiation of the production phase markedly promoted t-PA production. The volumetric productivity was stable for 30 days at 100 IU/cm³ per day. The cells were then mostly retained on microcarriers. Thus, an effective and scalable method of t-PA production by normal fibroblast cells was developed. Offprint requests to: S. Mitsuda