Phase variation: genetic analysis of switching mutants

Site-specific inversion of a controlling element is responsible for flagellar phase transition in Salmonella. When a 900 bp DNA sequence is in one configuration, it allows the expression of the H2 gene, a structural gene which codes for the flagellar antigen. When it is in the opposite configuration, the H2 gene is not expressed. A hybrid λ phage containing the invertible control region and the adjacent H2 gene was constructed, and expression of the H2 gene was shown to be regulated by the orientation of the inversion region. Transposon Tn5 insertion derivatives of this hybrid phage were isolated and λH2::Tn5 mutants defective for inversion (H2 switching) were selected and characterized. Two classes of switching phenotypes were observed among the mutants—those which had slightly reduced frequencies of transition from expression of the H2 gene (H2 on) to nonexpression (H2 off) (intermediate class) and those in which the frequency of transition was reduced at least three orders of magnitude (null class). Physical mapping of the Tn5 insertion sites revealed that in all mutants the insertion was located inside the inversion region. Tn5 insertion sites in the null class of mutants defined a region of DNA including approximately 500 bp which was necessary for inversion. Genetic complementation tests showed that these λH2::Tn5 mutants could invert the H2 gene control element if the 500 bp region was introduced in the trans configuration. It is concluded that a gene is located inside the inversion segment and codes for a protein which is required for the inversion event. Furthermore, the two sites at which the crossover event occurred functioned in a cis configuration and were required for inversion. The presence of a gene which is involved in controlling site-specific recombination events may be a general feature of transposon-like elements.     

Protease-nexin: A cellular component that links thrombin and plasminogen activator and mediates their binding to cells

This report identifies a component of normal human fibroblasts that forms a covalent linkage with thrombin and urokinase (urinary plasminogen activator) and mediates most of the specific cellular binding of these proteases. This component, here named protease-nexin (PN), is both associated with the cell surface and released into the culture medium. In several ways PN resembles antithrombin III (AT3), a prominent inhibitor of thrombin in serum: PN links thrombin, probably via an ester bond; PN does not link thrombin blocked at its catalytic site serine; PN has a high-affinity heparin-binding site; and heparin greatly accelerates the rate of linkage between soluble PN and thrombin. Despite these similarities, PN and AT3 are distinct; they differ in size and are not immunologically cross-reactive. Whereas AT3 regulates the proteolytic activity of thrombin in serum, PN may regulate the activity of serine proteases at and near the cell surface.     

Solubility of carbon dioxide in lipid bilayer membranes and organic solvents

Partition coefficients of carbon dioxide into lipid bilayers (liposomes) and organic solvents were measured as a function of temperature. The molar partition coefficient of CO2 into liposomes of egg lecithin at 25 degrees C was 0.95 (ml CO2/ml lipid)/(ml CO2/ml saline). The addition of an equimolar amount of cholesterol to the egg lecithin decreased the partition coefficient by about 25%. The partition coefficients for CO2 into liposomes at 25 degrees C were lower than the partition coefficients into octanol (1.3), hexadecane (1.5) and olive oil (1.7). The results are discussed in terms of the solubility-diffusion model of non-electrolyte transport through lipid bilayer membranes.     

Prostaglandin E1 effects on resting and cholinergically stimulated lower esophageal sphincter pressure in cats

Intraluminal esophageal manometry with a sleeve catheter was used to compare the magnitude of decrease in lower esophageal spincter (LES) pressure produced by an arterial or venous infusion of prostaglandin E₁ in cats. Arterial PGE₁ produced significantly lower LES pressures than venous PGE₁ (p < 0.05). Maximal decrease of 75% in basal LES pressure occurred with an associated 15% decrease in systolic blood pressure. The site of action of PGE₁ in producing LES hypotension was studied by injection of either edrophonium, or bethanechol during the maximal PGE₁ effects. Bethanechol, which acts directly on sphincteric smooth muscle, produced an increase in LES pressure during both saline and PGE₁ infusion, while the increases in LES pressure seen with edrophonium during saline infusion were blocked during the PGE₁ infusion. From these studies, we conclude that PGE₁ produces LES hypotension in the cat by an inhibitory effect on the cholinergic pathway responsible for maintaining LES tone. These studies pharmacologically reproduce the LES pressure abnormality previously reported in the cat during acid-induced esophagitis and support the hypothesis that PGE₁ may be involved in the pathogenesis of acute acid-induced lower esophageal sphincter abnormalities.     

POLLEN MORPHOLOGY OF HAPLOPAPPUS AND RELATED GENERA (COMPOSITAE—ASTEREAE)

Pollen grains of Haplopappus and related genera in the subtribe Solidaginae from North and South America were examined by light and scanning electron microscopy. The grains are consistently tricolporate and echinate. Some genera can be distinguished by pollen size, spine length, and number of spine rows between colpi. Based on these characters, the divergence of Benitoa from other members of the subtribe, as indicated by its morphology and secondary chemistry, is supported. Additionally, the recently suggested absence of a close relationship between Pyrrocoma and Oonopsis is indicated by their contrasting pollen types. This study demonstrates the potential of pollen studies in distinguishing some taxonomic groups in the Astereae.     

Primary in vitro antibody response of rabbit lymphoid cells and T-B cell collaboration in the absence of detectable mitogens

To investigate whether the antibody response and T-B-cell collaboration in vitro can be obtained in the absence of mitogens, a method of obtaining an in vitro primary anti-sheep red blood cell antibody response by rabbit spleen and lymph node cells was developed. We used Marbrook culture vessels and a specially prepared medium containing 10% autologous serum and maintained at pH 7.4–7.6. The system was shown to be devoid of any polyclonal mitogens as assessed by [³H]thymidine incorporation and by direct examination for blast cells in stained smears. The primary response increased continuously over the 5-day cultivation period and only IgM but not IgG plaque-forming cells (PFC) were detected. In over 20 experiments, the response ranged from 357 ± 17 to 4425 ± 110 PFC/10⁷ cultured cells with a median stimulation index of 52. The spleen cells required less antigen than the lymph node cells and 2-mercaptoethanol inhibited the response of the spleen cells but not that of the lymph node cells. Lymphocytes were separated into highly pure T- and B-cell populations by negative selection using antibody-coated human erythrocytes to rosette either T or B cells and Ficoll-Hypaque centrifugation to remove rosetted cells. Upon cultivation, B cells alone gave a low IgM response, whereas B cells reconstituted with T cells gave a response similar to that obtained with unseparated lymphoid cells. We concluded that: (a) optimal conditions for obtaining primary in vitro antibody responses using rabbit spleen and lymph node cells were established, (b) T-B-cell collaboration was demonstrated in the rabbit primary antibody response to sheep erythrocytes, and (c) the primary antibody response in vitro and T-B-cell collaboration may occur in the absence of detectable polyclonal mitogens.     

Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures

Summary Cryostat sections from rat gracilis muscles were incubated with different biotinylated lectins: Con A (Concanavilin A), WGA (Wheat germ agglutinin), SBA (soybean agglutinin), GS I and GS II (Griffonia simplicifolia agglutinin), LCA (Lens culinaris agglutinin), PNA (peanut agglutinin) and PSA (Pisum sativum agglutinin). The sections were subsequently treated with alkaline phosphatase conjugated avidin. The lectin binding sites were visualized after incubation in substrate media containing: (1) 5-bromo-4-chloro indoxyl phosphate and Nitro Blue tetrazolium or copper sulphate; (2) naphthol AS-MX phosphate or naphthol AS-BI phosphate and various types of diazonium salts; (3) -naphthylphosphate and Fast Blue BB; (4) -glycerophosphate according to the method of Gomori. The results obtained with the alkaline phosphatase methods were compared with those seen with a streptavidin-horseradish peroxidase procedure. Several chromogen protocols for visualizing alkaline phosphatase activity showed differences in the ability to detect lectin binding sites. A sarcoplasmic reaction was evident for Con A, GS II, WGA, LCA, and PSA after incubation in the indoxyl phosphate medium. Sarcoplasmic reaction for GS II was also noticed after incubation with naphthol AS-MX Fast Blue BB and -glycerophosphate. The latter substrate also gave rise to a sarcoplasmic Con A reaction. With the indoxylphosphate tetrazolium salt method some muscle fibres showed a very strong intracellular reaction after incubation with Con A and GS II while the staining intensity was weak in other fibres. The same muscle fibres were stained with PAS. No sarcoplasmic reactions were observed with either naphthol phosphate media or with the diaminobenzidine peroxidase methods. Further, the staining of the muscle fibre periphery, connective tissue, and capillaries was intensified using the indoxyl method. The indoxylphosphate-tetrazolium salt method seems to be suitable for future investigations of lectin binding sites in muscle sections.     

Effect of methabenzthiazuron on growth and nitrogenase activity in Vicia faba

The effects of the herbicide methabenzthiazuron (175 and 220 g ha^-1) on vegetative and reproductive growth, nodulation and nitrogenase activity of Vicia faba were studied in the field under Mediterranean conditions. Nitrogenase activity of excised nodules was estimated using the acetylene reduction assay four times during the developmental period. Leaf area index, dry weight and nitrogen content of the different parts of the plants were measured. Methabenzthiazuron-treated plants showed an increase in nodulation, nitrogenase activity and vegetative growth at early pod fill. Methabenzthiazuron also caused an increase in leaf N content and fruits. These were transient effects found during early and mid pot fill. Nevertheless, plants treated with these sublethal doses of herbicide improved seed production and nitrogen content of seeds at harvest time. The stimulatory effect of methabenzthiazuron on N₂ fixation and vegetative growth seems not be related with the transient stimulatory effect on photosynthetic capacity, also caused by the herbicide, since the stimulatory effect on N₂ fixation was apparent during pod fill, when photosynthetic capacity declined and was not modified by methabenzthiazuron.