Immunoblotting of mite aeroallergens collected with an indoor Burkard air sampler

We examined the kinetics of airborne levels of mite allergen particles in a house by combined use of an indoor Burkard air sampler and immunoblotting. Airborne mite allergens collected on the Burkard sampling tape were transferred onto a nitrocellulose membrane, reacted with mouse monoclonal anti-mite allergen (Der pI) antibody, then treated with alkaline phosphatase conjugated anti-mouse IgG. Finally, the blotted allergen on the membrane was reacted with BCIP/NBT phosphatase, and purple spots visible by the naked eye were produced. The shape of the spots was observed under a microscope, and the spot area was measured by an image processor. This technique might be useful for analyzing the behavior of airborne allergen particles in indoor environments.     

Detection of dilute antigen in large sample volumes using antibody-coated polyester cloth

Summary A model antibody, goat anti-rabbit IgG antibody, was adsorbed onto a disk of polyester cloth and then fixed into a column apparatus. The macroporosity of the cloth allowed rapid immunoconcentration of a model antigen, rabbit IgG, by passing a large volume of the dilute antigen through the antibody-coated cloth. Such immunoconcentration permitted detection of the dilute antigen which otherwise would have gone undetected.     

Detection of human blood by cloth-based enzyme immunoassay of immunoglobulin G

A specific and sensitive assay for the detection of human blood was developed using polyester cloth coated with goat anti-human IgG antibody to capture human IgG, an abundant and stable protein in blood. The captured IgG was detected by the reaction between goat anti-human IgG antibody-peroxidase conjugate and a chromogenic peroxidase substrate. Because the assay is simple and rapid, and permits simultaneous analysis of multiple samples, it has the potential to be used as a forensic test for human blood.     

Formation of bioorganic compounds in aqueous solution induced by flames

Flames of flammable gases, when blown against a surface of an aqueous solution of organic compounds, were found to induce oxidation as well as other reactions in the solution. This reaction would be regarded as a new model for formation of bioorganic molecules in the primitive hydrosphere exposed to some radical-containing atmosphere.     

Fluorometric estimation of surface potential change associated with chemotactic stimulation in Tetrahymena pyriformis

For the quantitative estimation of surface potential change in intact cells a method was devised with the use of fluorescent probes, 8-anilino-1-naphthalenesulfonate (ANS) and N-phenyl-1-naphthylamine (NPN). Estimated values in liposomes were compared with changes in the zeta potential determined from electrophoresis. Both values agreed within the experimental variation, showing the usefulness of the method. The method was also applied to Tetrahymena pyriformis, which exhibits chemotaxis to various chemical stimuli. The surface potential change was observed when the cell was stimulated not only by inorganic salts but also by electrically neutral, hydrophobic compounds. The surface potential started to change in accordance with the depolarization of the membrane potential, except for the case of K⁺. Changes in the surface potential of T. pyriformis in response to Ca²⁺ and K⁺ were compared with those in the membrane potential. The quantitative contribution of the surface potential to cell depolarization associated with chemoreception is discussed.     

Cloning of a Phosphate-Regulated Hemolysin Gene (Phospholipase C) from Pseudomonas aeruginosa

Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa is a phosphate (P_i)-regulated extracellular protein which may be a significant virulence factor of this organism. The gene for this hemolytic enzyme was cloned on a 4.1-megadalton (Mdal) fragment from a BamHI digest of P. aeruginosa PAO1 genomic DNA and was inserted into the BamHI sites of the multicopy Escherichia coli(pBR322) and P. aeruginosa(pMW79) vectors. The E. coli and P. aeruginosa recombinant plasmids were designated pGV26 and pVB81, respectively. A restriction map of the 4.1-Mdal fragment from pGV26 was constructed, using double and single digestions with BamHI and EcoRI and several different restriction enzymes. Based on information from this map, a 2.4-Mdal BamHI/BglII fragment containing the gene for phospholipase C was subcloned to pBR322. The hybrid plasmids pGV26 and pVB81 direct the synthesis of enzymatically active phospholipase C, which is also hemolytic. The plasmid-directed synthesis of phospholipase C in E. coli or P. aeruginosa is not repressible by P_i as is the chromosomally directed synthesis in P. aeruginosa. Data are presented which suggest that the synthesis of phospholipase C from pGV26 and pVB81 is directed from the tetracycline resistance gene promoter. The level of enzyme activity produced by E. coli(pGV26) is slightly higher than the levels produced by P. aeruginosa(pMW79) under repressed conditions. In contrast, the levels produced by P. aeruginosa(pVB81) are at least 600-fold higher than the levels produced by P. aeruginosa(pMW79) under repressed conditions and approximately 20-fold higher than those produced by P. aeruginosa(pMW79) under derepressed conditions. The majority (85%) of the enzyme produced by E. coli(pGV26) remained cell associated, whereas >95% of the enzyme produced by P. aeruginosa(pVB81) was extracellular. Analysis of extracellular proteins from cultures of P. aeruginosa(pMW79) and P. aeruginosa(pVB81) by high-performance liquid chromotography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the phospholipase C gene was cloned intact, and it is likely that several additional genes were cloned on the 4.1-Mdal fragment of DNA. It was also found that some of these genes encode proteins which are the same molecular weight as some previously described P_i-repressible proteins of P. aeruginosa. The existence of a P_i regulon of P. aeruginosa is proposed. It is likely that one of these genes also regulates the level of pyocyanin production by P. aeruginosa and that one or more play a role in transport or binding of P_i. The availability of the hybrid plasmids described herein will be useful in further studies on the role of this hemolysin in the virulence of P. aeruginosa and in the study of the genetics and physiology of P_i-regulated proteins.     

Population changes and ranging behaviour of wild Japanese monkeys at Mt. Kawaradake in Kyushu,Japan

Population changes and home range utilization of the wild Japanese monkey at Mt. Kawaradake have been studied since 1972. Age compositions of this troop were obtained over a seven-year period. Troop size decreased from over 100 to 40 individuals as a result of a capture in 1974. The capture affected directly and indirectly the troop's range and population dynamics. The troop reduced its range size from 4.7 km² to 2.67 km² and changed its utilization pattern in relation to the decrease in size. After the capture, the troop used one particular area intensively, whereas the rhythmic nomadic pattern had been observed as before. This may have been caused by the decrease in the overall food requirement of the troop. The birth rate increased significantly after the capture. However, troop size did not increase because of the low recruitment rate for adult females and the high mortality of juveniles.