Differences in airway responsiveness to leukotriene D4 in allergic sheep with and without late bronchial responses

Allergic sheep respond to inhaled antigen with either acute and late bronchial obstructions (dual responders) or only acute bronchoconstriction (acute responders). In this study we tested the hypothesis that one factor which may distinguish between these two populations is the difference in sensitivity to a specific mediator of airway anaphylaxis, leukotriene (LT) D₄ (a major component of slow reacting substance of anaphylaxis). We postulated that if the hypothesis was correct than dual responders should demonstrate increased airway responses to inhaled LTD₄ and that this increased responsiveness should also be reflected by a more severe response to inhaled antigen. To test this we used animals from both groups with the same degree of non-specific airway responsiveness to carbachol and determined their airway responses to controlled inhlation challenges with synthetic LTD₄ and antigen. Airway responsiveness to carbachol was determined by measuring the change in specific lung resistance (SR_L) to increasing concentrations of carbachol aerosol, and then identifying, by linear interpolation, the provocative carbachol concentration which produced a 150% increase (PC₁₅₀) in SR_L. Airway responses to LTD₄, and antigen were determined by measuring the percentage change in SR_L after a controlled inhlation challenge with either aerosol. Airway responsiveness to carbachol was not different between the two groups. There was, however, a difference (p<0.05) in the airway response to the same dose of LTD₄ in the two groups. Dual responders showed a 297±72% increase in SR_L as compared to a 90±13% increase in SR_L in the acute responders. Dual responders also showed a greater immediate and more prolonged response to antigen than did acute responders. These results suggest that increased responsiveness to LTD₄ may be one factor which may distinguish dual responders from acute responders.     

Fusion of Sendai virions or reconstituted Sendai virus envelopes with liposomes or erythrocyte membranes lacking virus receptors

Incubation of intact Sendai virions or reconstituted Sendai virus envelopes with phosphatidylcholine/cholesterol liposomes at 37 degrees C results in virus-liposome fusion. Neither the liposome nor the virus content was released from the fusion product, indicating a nonleaky fusion process. Only liposomes possessing virus receptors, namely sialoglycolipids or sialoglycoproteins, became leaky upon interaction with Sendai virions. Fusion between the virus envelopes and phosphatidylcholine/cholesterol liposomes was absolutely dependent upon the presence of intact and active hemagglutinin/neuraminidase and fusion viral envelope glycoproteins. Fusion between Sendai virus envelopes and phosphatidylcholine/cholesterol liposomes lacking virus receptors was evident from the following results. Anti-Sendai virus antibody precipitated radiolabeled liposomes only after they had been incubated with fusogenic Sendai virions. Incubation of N-4-nitrobenzo-2-oxa-1,3-diazole-labeled fusogenic reconstituted Sendai virus particles with phosphatidylcholine/cholesterol liposomes resulted in fluorescence dequenching. Incubation of Tb3+-containing virus envelopes with phosphatidylcholine/cholesterol liposomes loaded with sodium dipicolinate resulted in the formation of the chelation complex Tb3+-dipicolinic acid, as was evident from fluorescence studies. Virus envelopes fuse efficiently also with neuraminidase/Pronase-treated erythrocyte membranes, i.e. virus receptor-depleted erythrocyte membranes, although fusion occurred only under hypotonic conditions.     

Suppression of defective-sporulation phenotypes by mutations in the major sigma factor gene (rpoD) of Bacillus subtilis

Summary Mutations (crsA47 and crsA4) in the major sigma factor gene (rpoD) of Bacillus subtilis RNA polymerase have been found to be powerful intergenic suppressors of spoOB, spoOE, spoOF, spoOK and spoIIG mutations. The crsA47 suppressor restores sporulation of spoOE, spoOF, spoOK and spoIIG mutants to levels near those of wild type bacteria and substantially improves the sporulation of a spoOB strain. The crsA mutations are shown to prevent the induction by aliphatic alcohols of SpoO phenocopies in wild type B. subtilis cells.     

Molecular analysis of transferable tetracycline resistance plasmids from Clostridium perfringens.

Conjugative tetracycline resistance plasmids from 15 Clostridium perfringens isolates from piggeries were analyzed by restriction endonuclease digestion and agarose gel electrophoresis. Seven isolates from one farm were found to carry a 47-kilobase pair (kb) plasmid, pJIR5, which had EcoRI, XbaI, and ClaI profiles that were identical to those of a previously characterized plasmid, pCW3. An isolate from a second farm was found to carry a plasmid, pJIR6, which also was indistinguishable from pCW3. Five additional isolates from a third farm carried a 67-kb plasmid, pJIR2, which had at least 29 kb of DNA in common with pCW3. Finally, two isolates from a fourth farm were found to carry a 50-kb plasmid pJIR4, which appeared to consist of an entire pCW3 molecule with a 3-kb insertion. Comparative restriction maps of pCW3, pJIR2, and pJIR4 that identified the regions of homology among these plasmids were constructed. We suggest that many conjugative tetracycline resistance plasmids in C. perfringens may contain a pCW3-like core.     

DNA-mediated gene transfer of a mutant regulatory subunit of cAMP-dependent protein kinase

We have used DNA-mediated gene transfer of genomic DNA to introduce into wild-type Chinese hamster ovary (CHO) cells a mutant gene that confers resistance to the growth inhibitory effect of cAMP. This dominant mutation in CHO cell line 10248 is responsible for an alteration in the RI subunit (RI*) of the type I cAMP-dependent protein kinase (Singh, T. J., Hochman, J., Verna, R., Chapman, M., Abraham, I., Pastan, I.H., and Gottesman, M.M. (1985) J. Biol. Chem. 260, 13927-13933). The transformant 11564 which was studied in detail, has the same characteristics as the original mutant 10248 including continued growth in medium containing 8-Br-cAMP, an increase in the Ka for cAMP activation of the kinase, a greatly reduced amount of type II protein kinase activity, an altered incorporation of the photoaffinity label 8-N3[32P]cAMP into the RI* subunit of PKI, and an absence of cAMP-dependent phosphorylation of a Mr = 52,000 protein in intact cells. In addition, analysis of the DNA of the transformant indicates the presence of an increased amount of DNA for the RI gene. These results are consistent with the transfer of a mutant gene for the RI* subunit of the cAMP-dependent protein kinase and its phenotypic expression in the transformant and also support the hypothesis that the mutation responsible for the defect in cell line 10248 is due to an alteration in the gene for RI.     

Fusion between Sendai virus envelopes and biological membranes. The use of fluorescent probes for quantitative estimation of virus-membrane fusion

The fluorescent probes, N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine and lissamine-rhodamine-B-sulfonylphosphatidylethanolamine, were inserted at the appropriate surface density into membranes of reconstituted Sendai virus envelopes, thus allowing transfer of energy between the fluorescent probes. In addition, only the fluorescent molecule N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine was inserted into the viral envelopes, resulting in self-quenching. Incubation of fluorescent, reconstituted Sendai virus envelopes with human erythrocyte ghosts resulted in either reduction in the efficiency of energy transfer or in fluorescence dequenching. No reduction in the efficiency of energy transfer or fluorescence dequenching was observed when fluorescent, reconstituted Sendai virus envelopes were incubated with glutaraldehyde-fixed or desialized human erythrocyte ghosts. Similarly, no change in the fluorescence value was observed when nonfusogenic, reconstituted Sendai virus envelopes were incubated with human erythrocyte ghosts. These results clearly show that reduction in the efficiency of energy transfer or dequenching is due to virus-membrane fusion and not to lipid-lipid exchange. Incubation of reconstituted Sendai virus envelopes, carrying inserted N-4-nitrobenzo-2-oxa-1,3-diazolephosphatidylethanolamine, with cultured cells also resulted in a significant and measurable dequenching. However, incubation of nonfusogenic, fluorescent reconstituted Sendai virus envelopes with hepatoma tissue culture cells also resulted in fluorescent dequenching, the degree of which was about 50% of that observed with fusogenic, fluorescent reconstituted viral envelopes. It is therefore possible that, in addition to virus-membrane fusion, endocytosis of fluorescent viral envelopes results in fluorescence dequenching as well.     

Some aspects of the natural history and food selection ofAvahi laniger

Two groups ofAvahi laniger were studied in the Forêt de Analamozoatra near Perinet in the eastern rainforest of Madagascar from August to October 1984. Overlap between the home ranges of neighbouring groups ofA. laniger was minimal. Group size ranged from one to four individuals with a median group size of two. In four out of ten groups a baby was born between August and September.A. laniger were most active after dusk and before dawn. They had an extended resting period around midnight. Their diet consisted mostly of leaves from at least 17 different plant species. They also ate flowers. Fruit eating was recorded twice. Leaves eaten had high contents of protein and sugar but did not contain alkaloids. The concentration of condensed tannins did not differ between food items and non-food items. There was no indication of competition with other prosimians that might explain their nocturnality.     

Horseradish peroxidase based bienzyme elctrode for isocitrate

Coimmobilized horseradish peroxidase and D-isocitrate dehydrogenase were fixed to an oxygen electrode to assemble a bienzyme electrode for isocitrate determination. The linear measuring range for isocitrate of the sensor is between 0.1 and 2.0 mmol. I^?1, the coefficient of variation (20 measurements) is 3.6%. 8 samples per hour can be assayed. With one sensor preparation 140 measurements can be carried out.     

Effects of histamine on bronchial artery blood flow and bronchomotor tone

The effects of aerosolized 5% histamine (10 breaths) on bronchial artery blood flow (Qbr), airflow resistance (RL), and pulmonary and systemic hemodynamics were studied in mechanically ventilated sheep anesthetized with pentobarbital sodium. Histamine increased mean Qbr and RL to 252 +/- 45 and 337 +/- 53% of base line, respectively. This effect was significantly different from base line for 30 min after challenge. The histamine-induced increase in RL was blocked by pretreatment with the histamine H1 receptor antagonist, chlorpheniramine, whereas the histamine-induced elevation in Qbr was prevented by the H2 antagonist, metiamide. Both responses were blocked only when both antagonists were present. Changes in Qbr were not directly associated with alterations in systemic and pulmonary hemodynamics or arterial blood gas composition. In vitro histamine caused a dose-dependent contraction of ovine bronchial artery strips that was prevented by H1 antagonist. The H2 agonist, impromidine, caused relaxation of precontracted arterial strips and was more potent and efficacious than histamine, whereas H1 agonists failed to elicit a relaxant response. Thus these findings indicate that histamine aerosol induces a vasodilation in the bronchial vascular bed; histamine has a direct effect on Qbr that is independent of alterations in RL, systemic and pulmonary hemodynamics, or arterial blood gas composition; and, histamine-induced bronchoconstriction is mediated predominantly by H1-receptors, whereas increased Qbr is controlled predominantly by H2-receptors, probably located in resistance vessels. This local effect of histamine on Qbr may have important implications in the pathophysiology of bronchial asthma and pulmonary edema.