Identification of novel peptide agonists from a random peptide library for a 5-oxo-ETE receptor, a receptor for bioactive lipids

A combinatorial peptide library contains an enormous combination of amino acid sequences and drug candidates, but an effective screening strategy to identify a variety of bioactive peptides has yet to be established. In this article, a random hexapeptide library was screened to identify novel peptide ligands for a 5-oxo-ETE receptor (OXER), which is a G-protein-coupled receptor for bioactive lipids, by using an OXER-Gi1alpha fusion protein. We successfully identified 2 hexapeptides-Ac-HMQLYF-NH2 and Ac-HMWLYF-NH(2)-that exhibited agonistic activity. Although the corresponding affinities were relatively low (EC50 values of 146 and 6.7 microM, respectively), the activities were confirmed by other independent cell-based assay methods, namely, intracellular calcium mobilization and cell chemotaxis. This study demonstrates that a combinatorial peptide library may be screened using a [35S]GTPgammaS binding assay with G-protein-coupled receptor (GPCR)-Galpha fusion proteins, in general, and that of peptide ligands can be obtained even for nonpeptide receptors.     

Liposomes destabilize tRNA during heat stress

Biomembranes play an important role in cellular response to heat stress. In this study, we focus on the interaction between liposomes and tRNA. Upon heat treatment we determined circular dichroism spectra of tRNA in presence of liposomes prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and cholesterol (Ch). To compare thermal stability, midpoint temperature (T_m) of tRNA was calculated from normalized θ₂₀₈. Addition of POPC/Ch liposomes decreased the T_m value of tRNA from 48°C to 38°C. We conclude that POPC/Ch liposomes interact with tRNA and destabilize its conformation under heat stress.     

Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.     

Cloning,heterologous expression,and localization of a novel crystal protein gene fromBacillus thuringiensis serovarjaponensis strain Buibui toxic to scarabaeid insects

RecombinantEscherichia coli strains harboring pAG1, pAG2, pKBB100, and pKBB101 were cloned by using antiserum constructed against 130-kDa crystal protein antigen ofBacillus thuringiensis serovarjaponensis strain Buibui. DNAs in the recombinant strains hybridized to the 26-base oligonucleotide probe corresponding to N-terminal amino acids of the 130-kDa crystal protein of strain Buibui. Cultures of the recombinant strains were toxic to larvae of the cupreous chafer,anomala cuprea. Furthermore, the production of the 130-kDa polypeptide was demonstrated in the cells harboring pAG1 and pAG2 by immunoblot analysis with antiserum against the 130-kDa crystal protein. Southern hybridization analysis showed that the 130-kDa crystal protein gene is located on the chromosomal DNA of strain Buibui. On the other hand, DNA probes derived fromcryIA(a) andcryIIIA genes did not hybridize to the DNA of strain Buibui.     

Some Characteristics of Membrane-Associated Protein Kinase in Lemna paucicostata

A protein kinase which phosphorylates histone was isolated fromthe endoplasmic reticulum-rich fractions of Lemna paucicostata.The enzyme could be solubilized by sonication, and its molecularweight was estimated as 220,000 by Sephacryl S-300 gel filtration.The optimum pH for enzyme activity was 9.0–9.5 and theactivity was stimulated by Co^2$, Mg^2$ and Mn^2$. Substrate proteinswhich might be phosphorylated by this protein kinase were alsodetected in microsomal fractions of Lemna plants. ¹ Present address: Advanced Research Laboratory, HITACHI LTD.,Kokubunji, Tokyo 185, Japan.     

A Step in the Biosynthesis of Gibberellins that Is Controlled by the Mutation in the Semi-Dwarf Rice Cultivar Tan-Ginbozu

Genetic analysis and a comparison of endogenous levels of gibberellinsbetween the semi-dwarf rice cultivar Tan-ginbozu and the correspondingnormal cultivar Ginbozu have confirmed that Tan-ginbozu is agibberellin deficient mutant and that the semi-dwarfism of Tan-ginbozuis controlled by a single recessive gene. A step in the biosynthesisof gibberellins that is blocked by the mutation in Tan-ginbozuhad been considered to be the synthesis of ent-kaurene or anearlier step. However, the rate of production of ent-kaureneby Tan-ginbozu was almost the same as that by Ginbozu. By contrast,accumulation of only a small amount of ent-kaurene was detectedin Tan-ginbozu, and the amount that accumulated was similarto that in Ginbozu that had been treated with 6.9 x 10^-8 M uniconazole-P(an effective inhibitor of three oxidative steps in the pathwayfrom ent-kaurene to ent-kaurenoic acid via entkaurenol and ent-kaurenal).The height of the treated Ginbozu plants was reduced to thesame as that of Tanginbozu plants. Resembling Tan-ginbozu plants,Ginbozu plants that had been treated with uniconazole-P respondedwell to ent-kaurenoic acid and slightly to ent-kaurene and ent-kaurenol.Since the growth-promoting activity of enf-kaurenal in Tan-ginbozuwas similar to that of ent-kaurene, our results suggest thatthe mutation in Tan-ginbozu blocks the three oxidative stepswhereby ent-kaurene is converted to ent-kaurenoic acid. (Received June 9, 1995; Accepted February 15, 1996)     

Gravitropic response inEichhornia cressipes (water hyacinth) I. process of gravitropic bending in the peduncle

In the last half of the anthokinetic cycle inEichhornia cressipes the peduncle showed a two-step bending response which started after the completion of the flowering: the primary bending in the upper region and the second one in the base of the peduncle. In the present study, only the primary bending response was examined and the following results were obtained.

1. Under the condition in the Western Japan, the anthokinetic cycle completed in 36–40 hr. The inflorescence started to grow during night, reaching the full flowering stage next morning, and the bending was initiated in the evening and completed next morning.
2. The bending was a positive gravitropism since the peduncle did not show bending when it was placed on a horizontal clinostat rotating at 2–3 rpm, or when the plant was placed up-side-down.
3. Before the end of flowering phase, the peduncle showed a normal negative gravitropic response but afterward it acquired the property to show a positive gravitropic response.
4. The bending was due to the extension of convex side of the peduncle, accompanied by a shrinkage of the concave side. The extension of the upper side was caused by cell extension. At the turning point from negative to positive gravitropic response, the extension of the peduncle ceased for several hours.

From the above results it is concluded that the primary bending response of the peduncle in water hyacinth was a positive gravitropism.     

α-Galactosidase gene mutations in Fabry disease: heterogeneous expressions of mutant enzyme proteins

Five point mutations were identified in unrelated Japanese Fabry disease hemizygotes: three new missense mutations, C142Y (⁴²⁵ G A), A156V (⁴⁶⁷ C T), and L166V (⁴⁹⁶ C G) in exon 3; one new splice site mutation at the 3 end of the consensus sequence in exon 4; one previously reported nonsense mutation, W44X (¹³¹ G A). C142Y expressed 50% of the normal enzyme protein in COS-1 cells, but catalytic activity was not detected. Both A156V and L166V expressed significant amounts of residual enzyme activity (6.7% and 9.8%) and enzyme proteins (10% each), the latter were more thermolabile at neutral pH than at acid pH, in vitro.     

Occurrence of jasmonic acid in Dunaliella (Dunaliellales, Chlorophyta)

The occurrence of jasmonic acid and related compounds in Dunaliella species was investigated using gas-liquid chromatography/mass spectroscopy (GCY MS). Jasmonic acid was identified in the ethyl acetate soluble-acidic fraction of Dunaliella tertiolecta and Dunaliella salina (Dunal) Teodoresco, The concentration of jasmonic acid in D. salina. which is extremely halophilic, was much higher than that in D. tertiolecta Butcher, These results indicate that jasmonic acid might play an important role in salt-tolerance in Dunaliella.