Some properties of salivary amylases of Adelphocoris suturalis (Miridae), Dolycoris baccarum (Pentatomidae), and several other heteropteran species

Some properties of salivary amylases of the adults of Adelphocoris suturalis Jakovlev and Dolycoris baccarum L. were studied in vitro and compared with those of several other heteropteran species. The reducing sugar produced by the action of the salivary amylase of D. baccarum increased in proportion to substrate concentration while the concentration was relatively low (below 0.67% in its final concentration). Its increase stopped at the concentrations from 0.67 to 2.0%, and then it increased again constantly but slowly. The optimum temperature for the action of the enzyme was found to be about 50° (in A. suturalis) and about 40° (in D. baccarum), and the optimal pH was 3.5–4.0 in A. suturalis and 6.0 in D. baccarum. The salivary amylase activity of D. baccarum was scarcely affected by NaCl, KNO₃, and other compounds, while the salivary amylase of A. suturalis was strongly activated by NaCl and moderately by KNO₃. It seems highly probable that taxonomically related heteropterous insects might well be grouped with respect to the degree of activation of the salivary amylase by Cl- or NO₃-.

---

Zusammenfassung Einige Eigenschaften der Speichelamylase von Adelphocoris suturalis und Dolycoris baccarum wurden im Reagenzglas studiert und mit denen anderer Heteropterenarten verglichen. Die durch die Wirkung der Speichelamylase von D. baccarum produzierten Stärkehydrolysate vermehrten sich im Verhältnis zur Substratkonzentration bei einer verhältnismäßig niedrigen Konzentration (weniger als 0,67% in der Schlußkonzentration). Diese Vermehrung wurde bei einer Konzentration von etwa 0,67 bis 2,0 unterbrochen, um sich dann wieder stetig, aber langsamer fortzusetzen. Die optimale Temperatur für die Wirkung des Fermentes lag für A. suturalis bei etwa 50° und für D. baccarum bei etwa 40°. Die optimale Wasserstoff-Ionenkonzentration betrug für A. suturalis pH 3,5–4,0 und für D. baccarum pH 6,0. Die Aktivität der Speichelamylase von D. baccarum wurde durch NaCl, KNO₃ und andere Verbindungen kaum beeinflußt, während die Speichelamylase von A. suturalis durch NaCl stark und durch KNO₃ schwach aktiviert wurde. Es wird vermutet, daß, hinsichtlich des Aktivierungsgrades der Speichelamylase durch Cl- und NO₃-, systematisch nahe verwandte Heteropterenarten der gleichen Gruppe angehören.

---

---

Contribution No. 28 from the Laboratory of Entomology, Obihiro Zootechnical University.     

Two kinds of thick filament in smooth muscle cells in the adductor of a clam, Chlamys nobilis

The ultrastructure of the opaque portion of the adductor muscle in the pecten Chlamys nobilis was investigated. The opaque portion was composed of smooth muscle cells that contained thin and thick filaments. The thick filaments were classified into two kinds, thinner and thicker, according to the statistical analysis of diameters. They were also classified as being shorter and longer, when isolated native filaments were examined. The thick filaments may consequently be classified into two kinds: thinner and shorter filaments, and thicker and longer ones. The thinner and shorter filaments were about 26.5 nm in diameter and 7.5 mum in length, and the thicker and longer ones were about 42.0 nm in diameter and 13.0 mum in length, respectively. A regular periodicity was apparent on the surface of the core after removal of myosin molecules from its surface. The periodicity seemed similar for the two kinds of thick filament.     

Purification and properties of Renilla reniformis luciferase.

Luciferase from the anthozoan coelenterate Renilla reniformis (Renilla luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5.) catalyzes the bioluminescent oxidation of Renilla luciferin producing light (lambdaB 480 nm, QB 5.5%), oxyluciferin, and CO2 (Hori, K., Wampler, J.E., Matthews, J.C., and Cormier, M.J. (1973), Biochemistry 12, 4463). Using a combination of ion-exchange, molecular-sieve, sulfhydryl-exchange, and affinity chromatography, luciferase has been purified, approximately 12 000-fold with 24% recovery, to homogeneity as judged by analysis with disc and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and ultracentrifugation. Renilla luciferase is active as a nearly spherical single polypeptide chain monomer of 3.5 X 10(4) daltons having a specific activity of 1.8 X 10(15) hp s-1 mg-1 and a turnover number of 111 mumol min-1 mumol-1 of enzyme. This enzyme has a high content of aromatic and hydrophobic amino acids such that it has an epsilon280nm 0.1% of 2.1 and an average hydrophobicity of 1200 cal residue-1. The high average hydrophobicity of luciferase, which places it among the more hydrophobic proteins reported, is believed to account, at least in part, for its tendency to self-associate forming inactive dimers and higher molecular weight species.     

Mapping multiple disease resistance genes using a barley mapping population evaluated in Peru, Mexico, and the USA

We used a well-characterized barley mapping population (BCD 47 × Baronesse) to determine if barley stripe rust (BSR) resistance quantitative trait loci (QTL) mapped in Mexico and the USA were effective against a reported new race in Peru. Essentially the same resistance QTL were detected using data from each of the three environments, indicating that these resistance alleles are effective against the spectrum of naturally occurring races at these sites. In addition to the mapping population, we evaluated a germplasm array consisting of lines with different numbers of mapped BSR resistance alleles. A higher BSR disease severity on CI10587, which has a single qualitative resistance gene, in Peru versus Mexico suggests there are differences in pathogen virulence between the two locations. Confirmation of a new race in Peru will require characterization using a standard set of differentials, an experiment that is underway. The highest levels of resistance in Peru were observed when the qualitative resistance gene was pyramided with quantitative resistance alleles. We also used the mapping population to locate QTL conferring resistance to barley leaf rust and barley powdery mildew. For mildew, we identified resistance QTL under field conditions in Peru that are distinct from the Mla resistance that we mapped using specific isolates under controlled conditions. These results demonstrate the long-term utility of a reference mapping population and a well-characterized germplasm array for locating and validating genes conferring quantitative and qualitative resistance to multiple pathogens.     

Impact of Histone H1 on the Progression of Allergic Rhinitis and Its Suppression by Neutralizing Antibody in Mice

Nuclear antigens are known to trigger off innate and adaptive immune responses. Recent studies have found that the complex of nucleic acids and core histones that are derived from damaged cells may regulate allergic responses. However, no fundamental study has been performed concerning the role of linker histone H1 in mast cell-mediated type I hyperreactivity. In this study, we explored the impact of histone H1 on mast cell-mediated allergic responses both in vitro and in vivo. In the course of a bona-fide experimental allergen sensitization model upon co-injection with alum adjuvant, ovalbumin (OVA), but not PBS, induced elevated levels of circulating histone H1. Intranasal challenge with histone H1 to OVA/alum- (but not PBS/alum)-sensitized mice induced significantly severer symptoms of allergic rhinitis than those in mice sensitized and challenged with OVA. A monoclonal antibody against histone H1 not only suppressed mast cell degranulation, but also ameliorated OVA-induced nasal hyperreactivity and IgE-mediated passive cutaneous anaphylaxis. Our present data suggest that nuclear histone H1 represents an alarmin-like endogenous mediator acting on mast cells, and that its blockage has a therapeutic potential for mast cell-mediated type I hyperreactivity.     

Systematic separation and purification of elastase, gelatinase (matrix metalloproteinase 9), and collagenase (matrix metalloproteinase 8) from polymorphonuclear leukocytes in dialyzers previously used by patients with renal failure

We developed a simple and effective method for the systematic separation and purification of human polymorphonuclear leukocyte (PMN) proteinases, elastase, gelatinase (matrix metalloproteinase 9, type IV collagenase), and collagenase (matrix metalloproteinase 8), derived from the extracts of hollow fiber dialyzers that had been utilized in the treatment of patients with renal failure. The fraction containing elastase was grossly separated from that containing gelatinase and collagenase by heparin-Sepharose chromatography and purified in an aprotinin column. The remaining two enzymes were then separated using the gelatin-Sepharose column after gel chromatography following ammonium sulfate precipitation. Gelatinase and collagenase were further purified by gelatin-Sepharose chromatography as a latent form and by collagen-Sepharose chromatography as an activated form. This novel method offers procedural advantages over existing methods that separate PMNs from the whole blood of volunteers for experimental research purposes.