Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin.

We have previously shown that replacing the P1-site residue (Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lys results in the acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively. However, the inhibitory activities thus induced are not strong. In the present study, we introduced additional amino acid replacements around the reactive site to try to make the P1-site mutants more effective inhibitors of chymotrypsin or trypsin. The amino acid replacement Asp-->Tyr at the P2' site of OMCHI3(P1Met) resulted in conversion to a 35000-fold more effective inhibitor of chymotrypsin with an inhibitor constant (K(i)) of 1. 17x10(-11) M. The K(i) value of OMCHI3(P1Met, P2'Ala) indicated that the effect on the interaction with chymotrypsin of removing a negative charge from the P2' site was greater than that of introducing an aromatic ring. Similarly, enhanced inhibition of trypsin was observed when the Asp-->Tyr replacement was introduced into the P2' site of OMCHI3(P1Lys). Two additional replacements, Asp-->Ala at the P4 site and Arg-->Ala at the P3' site, made the mutant a more effective inhibitor of trypsin with a K(i) value of 1. 44x10(-9) M. By contrast, Arg-->Ala replacement at the P3' site of OMCHI3(P1Met, P2'Tyr) resulted in a greatly reduced inhibition of chymotrypsin, and Asp-->Ala replacement at the P4 site produced only a small change when compared with a natural variant of OMCHI3. These results clearly indicate that not only the P1-site residue but also the characteristics, particularly the electrostatic properties, of the amino acid residues around the reactive site of the protease inhibitor determine the strength of its interactions with proteases. Furthermore, amino acids with different characteristics are required around the reactive site for strong inhibition of chymotrypsin and trypsin.     

The reproductive strategy of the gregarious parasitoid,Pteromalus puparum (Hymenoptera: Pteromalidae)

Summary Pteromalus puparum is a gregarious parasitoid of many butterfly pupae. Adult size, mortality, and sex ratio of P. puparum, as a parasitoid of Papilio xuthus, were unit weight of the host. Effects of female size on fecundity, wing load, and longevity were also examined.The highest total weight of progeny from the host was attained when the number of eggs per gram of the host was approximately 150. Positive correlations were observed between the size of the females and their fecundity and wing load. The maximum longevity of the female kept with honey but without hosts was attained when the initial number of parasitoids per g of the host was 150.Considering the total fecundity of all female progeny, the reproductively most efficient number of eggs to be deposited per g of the host was estimated to be approximately 300. However, as shortage of food for the adult females strongly affects their fecundity, the reproductively most efficient number of eggs to be deposited per g of the host was about 70 when the adult female progeny was not provided with food.The optimal number of eggs to be deposited when the emale oviposits in the host under field conditions is discussed.     

Role of actin in the cAMP-dependent activation of sodium/glucose cotransporter in renal epithelial cells

We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin-Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [¹⁴C]-α-methyl glucopyranoside ([¹⁴C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [¹⁴C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments.     

Cleavage of double helical DNA by Cu2+ ion in the presence of bisintercalator containing penta(ethylene glycol) connector chain

In designing new DNA recognizing and cleaving reagents, we introduce herein a bisacridine derivative (referred to as bisacridine) in which two acridine heterocycles are connected by a penta(ethylene glycol) bridging chain. This compound offers two possible functions: 1, stabilization of DNA bisacridine intercalator complex by metal ion. The penta(ethylene glycol) chain stabilizes metal ions binding to the phosphate site of DNA, where the penta(ethylene glycol) chain constitutes a part of a pseudomacrocyclic ligand for metal binding; and 2, enhancement of metal-assisted hydrolytic cleavage of DNA by means of a metal concentration effect by the pseudomacrocyclic ethereal chain. The binding isotherms of bisacridine with DNA in the presence of metal ions showed that the binding was mainly governed by the cation exchange reaction on the anionic DNA polymer chain, i.e., the exchange between metal ions and the cationic bisacridine. The bisacridine showed an increase DNA binding ability compared to quinacrine, the monoacridine counterpart, and caused an enhancement of DNA cleavage in the presence of Cu2+ ions. Additional experiments which included DNase 1 footprinting in the presence of bisacridine and the DNA cleavage by Cu2+/bisacridine using a 32P end-labelled DNA fragment, suggested that the Cu2(+)-assisted DNA cleavage sites in the presence of bisacridine were in reasonable overlap with the DNA binding sites of bisacridine.     

Purification of a fibrolast-inhibitory factor from Actinobacillus actinomycetemcomitans Y4

Abstract A factor showing inhibitory activity against human gingival fibrolasts was extracted from the cytosol fraction of Actinobacillus actinomycetemcomitans Y4. The activity markedly inhibited the proliferation of human gingival fibrolasts, but had no effect on cell viability or gross morphology. No such activity was found in cytosol fractions from either Porphyromonas gingivalis 381 or Escherichia coli HB101. The extract from A. actinomycetemcomitans Y4 was then purified by anion-exchange chromatography, hydroxyapatite chromatography and gel-filtration chromatography to give a single band on SDS-PAGE with an apparent molecular mass of 65 kDa. The purification ratio was 183-fold with a recovery rate of 5% compared with the crude extract (starting material) when the activity was assessed by direct cell counts.     

New PCR-RFLP analysis for the mouse Tnfsf6gld gene caused by a point mutation in the Tnfsf6 [tumor necrosis factor (ligand) superfamily, member 6] locus.

A new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of the mouse Tnfsf6gld mutation. An artificial restriction site was introduced to the mouse Tnfsf6gld mutation by PCR amplification using a modified primer. The three genotypes of the Tnfsf6 locus (Tnfsf6gld/Tnfsf6gld, Tnfsf6gld/+, and +Tnfsf6-gld/+Tnfsf6-gld) could be distinguished clearly and easily. This PCR-RFLP analysis was found to be useful for the identification of the mouse Tnfsf6gld mutation.     

Kyotorphin (tyrosine-arginine) synthetase in rat brain synaptosomes

Kyotorphin (Tyr-Arg) is a unique neuropeptide which produces analgesia by releasing Met-enkephalin from slices of the brain and spinal cord. Recent studies revealed that kyotorphin possesses the properties of neurotransmitter/neuroregulator. In the present study, we identified a kyotorphin synthetase in the soluble fraction of rat brain synaptosomes (synaptosol) and characterized it. The enzyme partially purified with Sephacryl S-300 showed an absolute requirement for ATP, MgCl2, tyrosine, and arginine. The optimal pH was 7.5-9.0 and the pI was determined to be 6.1-6.2 by isoelectric focusing. The Km was 25.6 microM for tyrosine, 926 microM for arginine, 294 microM for ATP, and 442 microM for MgCl2. The Vmax was 34.0 pmol/mg of protein/h. The apparent molecular size of this "kyotorphin synthetase" further purified by the DE52 column was 240,000-245,000 daltons, estimated using TSKgel G4000SW column chromatography. The enzyme reaction is represented by the following equation: Tyr + Arg + ATP + MgCl2 + kyotorphin synthetase----Tyr-Arg (kyotorphin) + AMP + PPi + MgCl2 + kyotorphin synthetase. The regional distribution and subcellular localization of the synthetase showed a close correlation to that of kyotorphin levels in the rat brain. The amounts of kyotorphin formed from amino acids by the synthetase in the dialyzed synaptosol was 3.0-4.0 times higher than that from precursor proteins by processing enzymes within the 30 min incubation.     

The role of phospholipase in beta-agonist-induced down regulation in guinea pig lungs

It has been observed that repeated and prolonged beta-agonist treatment causes the impairment of beta-adrenergic function, so-called "desensitization" or "down regulation". To clarify the mechanism of down regulation, the following experiment was performed using guinea pig lungs. Animals were divided into four groups: In the metaproterenol groups, guinea pigs were treated with metaproterenol (10 mg/kg/day) by intraperitoneal injection once a day for 1 day or for 7 successive days In the control groups, guinea pigs were treated with saline by the same procedure as in the metaproterenol groups. In the group treated with metaproterenol for 7 days, there was a 45% reduction in the number of beta-adrenoceptors and a 62% reduction in adenylate cyclase activity, compared with those of the control group. However, there were no significant changes in the dissociation constant (Kd) of the receptors. On the other hand, no reduction in the number of beta-adrenoceptors and adenylate cyclase activity was observed in the group treated with metaproterenol once a day for 1 day, compared with those of the control group. Phospholipase (PLase) activity in the lung microsomes of guinea pigs injected with metaproterenol for 1 day and for 7 days was elevated by 14.4 and 33.1%, respectively, compared with that of the control groups. Phospholipid contents of lung membranes prepared from the animals treated with metaproterenol for 7 days were significantly decreased compared with those of the control group, though in the group treated with metaproterenol once a day for 1 day, phospholipid contents did not differ from those of the control. Lung membranes treated with PLase A2 revealed decreases both in the number of beta-adrenoceptors and adenylate cyclase activity, dose dependently. These results and the fact that membrane phospholipids are involved in the beta-adrenoceptor system suggest that down regulation observed during beta-agonist administration is, at least in part, attributed to degradation of phospholipids of lung membranes by the persistent activation of PLase in the tissue.