Novel and sensitive noncompetitive enzyme immunoassay for kassinin in rat plasma

Summary A novel and sensitive noncompetitive enzyme immunoassay for kassinin is described. Kassinin was biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate. The biotinylated kassinin was trapped on antikassinin IgG-coated polystyrene balls and, after washing to eliminate other biotinylated substances, was eluted with HCl. The biotinylated kassinin eluted was reacted with anti-kassinin Fab'-peroxidase conjugate and trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry. The detection limit of kassinin was 0.13 pg (0.1 fmol)/tube or 0.065 g/l of rat plasma, which was 750-fold or 15-fold lower than that for competitive radioimmunoassay.     

Development of a new tachykinin antagonist

The mouse urinary bladder possesses a tachykinin receptor which responds to kassinin and eledoisin, but not to some other tachykinins. The action of kassinin, but not that of eledoisin, was blocked in a surmountable manner by a new tachykinin antagonist, D-Pro4, Val8, D-Trp (SP).     

Development of a new tachykinin antagonist

Summary The mouse urinary bladder possesses a tachykinin receptor which responds to kassinin and eledoisin, but not to some other tachykinins. The action of kassinin, but not that of eledoisin, was blocked in a surmountable manner by a new tachykinin antagonist, D-Pro⁴, Val⁸, D-Trp^7,9,10 (SP)^4–11.Acknowledgments. We thank Mrs Danielle Solomos for technical help. This study was financed by a grant from the Swiss National Science Foundation (3.800-80.2).     

Bioassay for hamster macrophage chemotaxis: application to study particle-lung interactions

Attraction of lung macrophages to particle deposition sites has been demonstrated in different animal species. We reported a threefold increase of the number of macrophages to occur within 40 min after polystyrene particle deposition in hamster airways [Geiser et al. (1994) Am. J. Respir. Cell Mol. Biol. 160: 594–603]. Complement-derived chemotactic activity is one of the mechanisms postulated for macrophage recruitment. It was the aim of this study to test whether complement-derived chemotactic activity is involved in the rapid recruitment of macrophages to the site of deposited polystyrene particles in hamster airways. We first developed an in vitro cell migration assay for hamster macrophages to assess complement-derived chemotaxis. Second, the bronchoalveolar lavage fluids (BALF) of four hamsters that had inhaled aerosols of polystyrene microspheres were tested for chemotactic activity by this bioassay and compared with BALF of four sham-exposed hamsters. Chemotactic response of macrophages was found toward complement-activated hamster serum, whereas macrophage migration was not increased toward BALF of particle and sham-exposed hamsters. In contrast, macrophage migration to BALF of both groups was reduced by 1.6-fold. Thus, the stimulus for macrophage recruitment to the site of deposited polystyrene particles in hamster airways could not be demonstrated using this bioassay. Received 10 September 1997; received after revision 24 November 1997; accepted 10 December 1997     

Amino acid composition and sequence of kassinin,a tachykinin dodecapeptide from the skin of the African frog Kassina senegalensis

Summary Methanol extracts of the skin of the African amphibian Kassina senegalensis contain a dodecapeptide, kassinin, belonging to the family of tachykinins or physalaemin-like peptides. Kassinin, like all other natural tachykinins, possesses the characteristic C-terminal tripeptide Gly-Leu-Met-NH₂ and a phenylalanine residue in position 5 from the C-terminus. However, the amino acid sequence in the N-moiety of the molecule differs sharply from that of the other tachykinins.Supported in part by grants from the Consiglio Nazionale delle Ricerche, Roma.     

Further studies on gel filtration of urinary substances

Summary Urine specimens from healthy children were concentrated and filtered through Sephadex G-100 and G-25. The eluate was recovered in fractions of 5 ml each. Total hexoses, galactose, total proteins and total uronic acids were determined on each fraction. Total hexoses and galactose were eluted in 6 distinct peaks. The uronic acid containing GAG were eluted in 4 small peaks on G-25 and 1 peak on G-100. This peak is eluted always with high amounts of galactose containing glycoproteins.This work was partially supported by research grants from the CONICET, República Argentina.     

Further studies on gel filtration of urinary substances.

Urine specimens from healthy children were concentrated and filtered through Sephadex G-100 and G-25. The eluate was recovered in fractions of 5 ml each. Total hexoses, galactose, total proteins and total uronic acids were determined on each fraction. Total hexoses and galactose were eluted in 6 distinct peaks. The uronic acid containing GAG were eluted in 4 small peaks on G-25 and 1 peak on G-100. This peak is eluted always with high amounts of galactose containing glycoproteins.     

Isolation of a growth-stimulating agent from human skin fibroblast cultures

Summary Cell-free supernatants were harvested from cultures of human skin fibroblasts, were applied on to DEAE-cellulose columns, and the first fraction eluted with phosphate-buffered saline contained the growth-stimulating agent. The eluted fraction was then passed through a series of amicon membranes. After passing through PM-10, the filtrate stimulated growth of bovine vascular endothelial, canine myocardial, and human mammary carcinoma cells.     

Europe's Infrastructure Transition: Economy,War, Nature

In his many uses of the pendulum as a model for other motions, Galileo also described several of the properties of pendular motion. All but a small number of his apparently observational reports ring true because of his use of such qualifiers as ‘almost’. His report of observations of two lead balls on equal long strings is shown by reconstruction to have been a real experiment. His report of similar observations with balls of cork and lead is shown to be an imaginary experiment. His claim that the period of a pendulum is independent of amplitude is shown to be based more on mathematical deduction than on experimental observation.     

Separation of modulator-dependent protein kinase (type I) from cyclic GMP-dependent protein kinase in mouse testes

A new type of enzyme, modulator-dependent protein kinase (type I) (M-PKI), was successfully isolated from the cytosol fraction of mouse testes. It was eluted slightly after the peak of cyclic GMP-dependent protein kinase (G-PK) by Sephadex G-200 gel filtration. Unlike either cyclic AMP-dependent protein Kinase (A-PK) or G-PK, its maximal activity depended exclusively on the presence of crude protein kinase modulators (PKM) or partially purified stimulatory modulator (PKMs).     

Adhesion of human red blood cells to polystyrene. Influence of sodium chloride concentration and of neuraminidase treatment

Summary The adhesion on polystyrene of glutaraldehyde-fixed human red blood cells was found to increase with NaCl concentration. Half of the maximum of irreversible adhesion was obtained in 2.2 mM NaCl for neuraminidase-treated cells and in 5.5 mM NaCl for the untreated cells.     

Adhesion of human red blood cells to polystyrene. Influence of sodium chloride concentration and of neuraminidase treatment.

The adhesion on polystyrene of glutaraldehyde-fixed human red blood cells was found to increase with NaCl concentration. Half of the maximum of irreversible adhesion was obtained in 2.2 mM NaCl for neuraminidase-treated cells and in 5.5 mM NaCl for the untreated cells.     

Immunologic blood-brain barrier in the polyoma virus--Syrian hamster system]

The authors report premilinary results of an experiment on permeability of the blood-brain barrier (BBB) to anti-tumor virus-induced immunological factors in the polyoma virus/Syrian Hamster system. The animals were protected by subcutaneous or intracranial injections with virus before challenge with polyoma virus transformed cells by both routes. BBB seemed to be permeable to the efferent part of the subcutaneously induced immune reaction. On the contrary, antigenic information introduced in the central nervous system was trapped inside the BBB. Thus the BBB might offer a "one-way" permeability in this system.     

Modulation of signal transduction through the cellular prion protein is linked to its incorporation in lipid rafts

Because expressed at a significant level at the membrane of human T cells, we made the hypothesis that the cellular prion protein (PrP^c) could behave as a receptor, and be responsible for signal transduction. PrP^c engagement by specific antibodies was observed to induce an increase in cytosolic calcium concentration and led to enhanced activity of Src protein tyrosine kinases. Antibodies to CD4 and CD59 did not influence calcium fluxes or signaling. The effect was maximal after the formation of a network involving avidin and biotinylated antibody to PrP^c and was inhibited after raft disruption. PrP^c localization was not restricted to rafts in resting cells but engagement was a prerequisite for signaling induction, with concomitant PrP^c recruitment into rafts. These results suggest a role for PrP^c in signaling pathways, and show that lateral redistribution of the protein into rafts is important for subsequent signal transduction.Received 22 July 2004; received after revision 10 September 2004; accepted 7 October 2004     

Heterologous immunoadsorption in the purification of enzymes: N-acetyl-β-glucosaminidase

Summary An immunoadsorbent column was prepared using a specific antibody toN-acetyl--glucosaminidase of human origin. Although no precipitating activity of the antisera was found with mouse or rat liver extracts, enzyme was easily eluted from the column which provided about 50fold, single-step purifications of these heterologous enzymes.     

Analyses immunoélectrophorétiques des fractions protéiques du sérum humain séparées par électrophorèse en gel d'amidon

Summary Human plasma components, separated by starch gel electrophoresis, are eluted and concentrated by lyophilisation. Immuno-electrophoretic analysis shows that certain of these fractions are immunologically heterogenous. The authors point out that this immunological criterium should be used before employing an eluted fraction, even if it appears electrophoretically homogenous.      

Hemagglutination by simian adenovirus 7]

Simian adenovirus 7, either complete virus or its capsid subunits, agglutinates Rat (Sprague-Dowley) red blood cells in the presence of heterotypical antiserum. Haemagglutination takes place at 4 degrees C and room temperature. The antigen could not be eluted and its haemagglutinin properties are heat-stable. The reaction is specific. It is inhibited by homologous antiserum only. This property and its characteristics permit a camparison of this strongly oncogenic adenovirus to the human adenovirus of subgroup III of Rosen.     

Enhancement of fluorescence of pyrene-containing lipids by polar media, detergents and phospholipids

The fluorescence intensities of a medium-chain fatty acid and of several amphiphilic lipids, each containing pyrene in covalent linkage, were enhanced considerably by: 1) Dissolving in mixtures of a polar solvent (e.g. methanol, ethanol, tetrahydrofuran or dimethylsulfoxide) and water; for each individual compound, a certain ratio of solvent to water provided maximal fluorescence intensity. 2) Incorporating into micelles of reduced Triton X-100; an excess of detergent was used so that, statistically, only one molecule of lipid resided in one micelle of the Triton X-100. 3) Incorporating into liposomes of egg phosphatidylcholine; maximal fluorescence was observed using a large excess of phosphatidylcholine. When related to the fluorescence intensities in chloroform/methanol (2:1, by vol.) or water, the enhancement of fluorescence in the above three systems was about 2-6-fold or up to 60-fold, respectively.     

Bombesin-like immunoreactivity in the pancreas of man and other mammalian species

Summary Bombesin-like immunoreactivity has been measured in pancreatic tissues of man (12.4±1.2 pmol/g), pig (15.8±3.2), calf (4.3±0.9), rat (8.5±1.2) and guinea-pig (2.8±0.6) by a specific radioimmunoassay. Gel filtration of the pancreatic extracts revealed 2 major immunoreactive peaks: the earlier peak was eluted in the position of porcine gastrin-releasing peptide, and the later peak was eluted just after the amphibian bombesin standard. Immunocytochemistry demonstrated the presence of bombesin-like immunoreactivity in nerves in the rat pancreas, particularly in the exocrine pancreas, and occasionally in the peri-insular spaces. Isolated rat pancreatic islets were found to contain small quantities of bombesin-like immunoreactivity (0.037±0.003 fmol/islet) suggesting that mammalian bombesin-like peptides may be invovled in the regulation of endocrine as well as exocrine pancreatic secretion.Acknowledgments. We are grateful to Dr. A. V. Edwards, Physiological Laboratory, Cambridge, U. K. for providing the calf tissues, and the British Diabetic Association, U. K. for support.To whom reprint requests should be addressed.     

Enzyme immunometric assay for the determination of pregnancy associated plasma protein A (PAPP-A) with the antigen as solid phase (conjoint IEMA)

Purified pregnancy associated plasma protein A (PAPP-A) can be effectively bound to polystyrene microtitre plates. This immobilized antigen competes with the added serum PAPP-A of unknown concentration for the limited amount of peroxidase-labeled monospecific anti-PAPP-A antibody incubated simultaneously. The sensitivity is 0.1 WHO unit/ml and non-specific binding is 1.0%.