Oligosaccharides in the honeydew of Coccoidea scale insects: Coccus hesperidum L. and a new Stigmacoccus sp. in Brazil

Analysis of the honeydew from an as yet undescribed, though distinctive, Brazilian Stigmacoccus sp. (near S. asper Hempel) by paper chromatography, Fast atom bombardment (FAB-MS) and Gas chromatography-mass spectrometry (GC-MS) identified fructose and glucose as monosaccharides and sucrose, maltose, trehalulose, trehalose and a hexose-hexitol as disaccharides. Erlose and glucosyl erlose have been identified as the tri- and tetra-saccharides in Stigmacoccus sp. and characterised for the first time in scale insects by modern techniques of linkage analysis. The same erlose oligosaccharides were recognised in honeydew of the common scale insect Coccus hesperidum L., together with the pentamer of this series, maltosyl erlose, therefore recognising that specific metabolic transformations of sugars into this oligomeric series occur rather widely in Coccoidea scale insects.

Preliminary characterization of the multiple forms of ram sperm hyaluronidase

An investigation was made of the inter-relationships and characteristics of various hyaluronidase forms isolated from ram spermatozoa. They were shown to be members of an oligomeric series, apparently formed by intermolecular disulphide cross-linking. Two monomer species were detected, alpha (Mr 89,600) and beta (Mr 81,200). Although the alpha species predominated, the two were evenly distributed throughout the oligomer population, and they shared antigenic determinants; the beta species did not arise from the alpha species as a result of catabolism following cell disruption. The oligomeric series was of the form [Hyal]n, where n = 1, 2, 4, 5, 6, 7 etc.; no trimer was detectable. Though essentially cationic, part of the hyaluronidase population also had anionic characteristics, probably due to oxidation of free thiol groups. In the anionic subpopulation tetramers and higher oligomers predominated, whereas the non-anionic subpopulation was composed of monomers, dimers and tetramers. The pH optimum of the monomer was 4.3 in 0.2 M-NaCl/0.1 M-sodium citrate, whereas that of the anionic oligomers was 4.9. Both serum albumin and polylysine stimulated enzyme activity at pH 4.0 in the absence of NaCl; polylysine was particularly effective. NaCl diminished the stimulatory effects, and essentially suppressed them above the pH optimum. The specific activities of different oligomer populations were the same as that of the monomer, and conversion of oligomers into monomer by reduction had likewise no effect upon the specific activity. Low concentrations of poly(vinyl alcohol), poly(ethylene glycol) or polyvinylpyrrolidone stabilized soluble hyaluronidase activity by preventing the enzyme's binding to surfaces; solutions of anionic oligomers were further stabilized by NaCl. Enzyme preparations were stable for several months frozen in the presence of poly(vinyl alcohol) and salt.

Analytical Characterization of Tackifying Resins

Phenolic resins are added to rubber compounds to improve the tack (or autohesion) characteristics. These resins are generally oligomers of alkylated phenols and formaldehyde. Analytical characterizations of these resins are very limited and incomplete in the open literature. We have examined several types of resins using mass spectroscopy (principally field desorption, FD-MS), liquid chromatography (LC), and infrared spectroscopy (ATR-IR). Several oligomeric series were identified by FD-MS in t-octylphenol and t-dodecylphenol resins. ATR-IR analysis showed that the t-octyl groups are essentially completely p-substituted on the phenolic ring. LC was only of limited utility in obtaining analytical separations of resin oligomers. Higher molecular weight oligomers (≿1500) showed little if any LC resolution. Resins prepared under different manufacturing conditions showed additional series of oligomers. These were either “cyclic ethers” and/or amine-containing compounds, depending on the synthetic procedures used.

Inactivation of FVIII by Plasmin: Molecular Conformational Changes

Reduced SDS PAGE failed to detect proteolysis of the 200,000 subunit when purified FVIII was slowly inactivated by plasmin until less than 1% of the VIIIc was left. Graphs of VIIIc loss show highly repeatable discontinuities (8 expts.), suggesting a conformational change, as do transitorily increased then partially decreased VIII R:AG levels. The mobility of FVIII as judged by crossed-immunoelectrophoresis remained unchanged, but the area under the ‘arcs’ increased dramatically. Crossed-immunoelectrophoresis plates were washed, the ‘arcs’ cut out, melted and subjected to reduced SDS PAGE. Only the 200,000, IgG ‘H’ and ‘L’ bands were seen; the latter increased markedly in contrast to the 200,000 band. Also SDS denatured digestion aliquots were treated with a trace of mercaptoethanol and applied to running SDS PA gels. A rise, then fall in the 200,000 monomer:dimer ratio showed that during inactivation FVIII is more thoroughly ‘disorganised’ by SDS. 2.75 % PAGE (1) or SDS PAGE in similar gels (2) show that a linear relationship obtains between the log of the FVIII oligomer no. and their relative mobilities up to oligomers ‘8’ and ‘6’ respectively; above this new linear relationships obtain. When FVIII is briefly inactivated by a higher concentration of plasmin the ‘inflection point’ in (1) is lost and later reappears at ‘5’; in (2) it shifts to oligomer no. ‘3’ long after inactivation. These results show that during inactivation by plasmin FVIII undergoes a molecular ‘opening-out’ to reveal more antigenic sites before detectable breakdown of the 200,000 subunits and that changes occur in the native oligomeric series.

IMMUNOFLUORESCENT ANTIBODIES TO BOVINE KERATOHYALIN AND IMMUNOLOGIC CONFIRMATION OF HOMOLOGY

Extraction of bovine hoof epidermis with 1.0 M potassium phosphate buffer (pH 7.0) and dialysis of the extract against distilled water results in the formation of macroaggregates. Fractionation of solubilized macroaggregates on polyacrylamide gels reveals a 13-member oligomeric series. Indirect immunofluorescence with antisera from rabbits immunized with either macroaggregates or gel slices with individual oligomers demonstrates specific fluorescence over keratohyaline granules. The reaction is tissue-specific, but antibodies cross-react with goat, sheep, wildebeest and rabbit keratohyaline granules, although not against human, rat or guinea pig keratohyalin. Immunofluorescent methods show that the macroaggregates are a component of keratohyalin and that the 13 oligomers are antigenically identical.