Classification of Proteases in Antarctic Krill

The species of endogenous proteases in Antarctic krill, Euphausia superba, were investigated using the homogenate of krill and the active fractions after gel filtration of the homogenate as to the following criteria: Substrate specificity (benzyloxycarbonyl (Z)-Phe-Ala, Z-Glu-Tyr, hippuryl-Arg, hippuryl-Phe, benzoyl Arg-p-nitroanilide, Leu-p-nitroanilide, ¹⁴C-hemoglobin), sensitivity to protease inhibitors (diisopropyl fluorophosphate, iodoacetamide, EDTA, soybean trypsin inhibitor and pepstatin), molecular weight and isoelectric point.

From the experimental results, we found that the carboxypeptidase A and B, aminopeptidase, trypsin and cathepsin A types of proteases were present in krill.     

Effect of Hydrotropism on Root System Development in Soybean (Glycine max): Growth Experiments and a Model Simulation

To observe root system development, soybean plants (Glycine max) were grown in root boxes that were set horizontally to reduce the effect of gravity. Along with the root system development, the two-dimensional distribution of soil water content in the root boxes was measured continuously by the time domain reflectometry (TDR) method. Root system development and its morphological architecture were strongly affected by the positions of the water supply. It is suggested that root hydrotropism plays the dominant role in root system development. In addition to root hydrotropism, the importance of root compensatory growth is suggested. A combined model of root system development and soil water flow considering root hydrotropism and compensatory growth was used to simulate root system development and soil water flow. The morphological architecture of root systems and the distribution of soil water content obtained in the experiment were successfully explained by the model simulation. These results confirmed that root hydrotropism and compensatory growth are dominant factors in root system development under a reduced effect of gravity. The validity of the model was confirmed, and its applications for various purposes were suggested.     

Effect of an amyloidogenic sequence attached to yellow fluorescent protein

Green fluorescent protein (GFP) is often misfolded into nonfluorescent states when an aggregatable sequence is attached to its N-terminus. However, GFP fusions with highly aggregatable, prion-determining, and highly charged sequences from yeast prions, such as Sup35 and Ure2p, form green fibrils with properly folded GFP. To gain further insight into the general effect of an aggregatable sequence attached to fluorescent protein, we designed eight fusion proteins of a yellow variant of GFP (YFP) containing an aggregation-prone amyloidogenic sequence derived from human medin, attached via different lengths of linker sequence. Seven fusion proteins formed white fibrils lacking native YFP function. However, the fusion with an 18-residue medin sequence and a 50 amino acid linker formed fibrils with yellow color of folded YFP. Deconvolution analysis of infrared spectra also supports the presence of properly folded YFP in the fibrils formed by this protein. These results suggest that, the presence of an amyloidogenic sequence to a folded protein can promote the formation of fibrils and disrupt the native structures whereas the structure of the folded region is retained by optimizing sequences of amyloidogenic and linker regions.     

Molecular phylogeny of the Myzorhynchella Section of Anopheles (Nyssorhynchus) (Diptera: Culicidae): genetic support for recently described and resurrected species

Phylogenetic relationships among species of the Myzorhynchella Section of Anopheles (Nyssorhynchus) were investigated using the nuclear ribosomal DNA second internal transcribed spacer (ITS2), the nuclear whitegene and mitochondrial cytochrome oxidase subunit I (COI) regions. The recently described Anopheles pristinus and resurrected Anopheles guarani were also included in the study. Bayesian phylogenetic analyses found Anopheles parvus to be the most distantly related species within the Section, a finding that is consistent with morphology. An. pristinus and An. guarani were clearly resolved from Anopheles antunesi and Anopheles lutzii, respectively. An. lutzii collected in the same mountain range as the type locality were found within a strongly supported clade, whereas individuals from the southern state of Rio Grande do Sul, tentatively identified as An. lutzii based on adult female external morphology, were distinct from An. lutzii, An. antunesi and from each other, and may therefore represent two new sympatric species. A more detailed examination of An. lutzii sensu latoalong its known geographic range is recommended to resolve these anomalous relationships.     

Analysis of hydrolytic activity of a 65-kDa chitinase from the silkworm,Bombyx mori

The hydrolytic reactions of Bombyx mori 65-kDa chitinase with the short substrates, N-acetyl-chitooligosaccharides, were analyzed by HPLC. Analysis of the hydrolyzed products showed that the newly produced oligosaccharides are all beta anomers, suggesting that, similar to other family 18 glycosyl hydrolases, the 65-kDa chitinase acts in the retaining mechanism. Furthermore, the enzyme cleaves the N-acetylchitooligosaccharides mainly at the linkage between the second and the third GlcNAc moieties from the non-reducing end, while the other sites were cleaved in smaller proportions. Moreover, the initial reaction rates of the enzyme with the longer N-acetylchitooligosaccharides were higher than those with shorter ones. These results suggest that the enzyme is an endo-cleaving type and more efficient on the longer substrates.     

Development of an efficient maintenance and screening system for large-insert genomic DNA libraries of hexaploid wheat in a transformation-competent artificial chromosome (TAC) vector

Three large-insert genomic DNA libraries of common wheat, Triticum aestivum cv. Chinese Spring, were constructed in a newly developed transformation-competent artificial chromosome (TAC) vector, pYLTAC17, which accepts and maintains large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. The vector contains the cis sequence required for Agrobacterium-mediated gene transfer into grasses. The average insert sizes of the three genomic libraries were approximately 46, 65 and 120 kbp, covering three haploid genome equivalents. Genomic libraries were stored as frozen cultures in a 96-well format, each well containing approximately 300-600 colonies (12 plates for small library, four for medium-size library and four for large library). In each of the libraries, approximately 80% of the colonies harbored genomic DNA inserts of >50 kbp. TAC clones containing gene(s) of interest were identified by the pooled PCR technique. Once the target TAC clones were isolated, they could be immediately transferred into grass genomes with the Agrobacterium system. Five clones containing the thionin type I genes (single copy per genome), corresponding to each of the three genomes (A, B and D), were successfully selected by the pooled PCR method, in addition to an STS marker (aWG464; single copy per genome) and CAB (a multigene family). TAC libraries constructed as described here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning.     

Aqueous flare elevation induced by transcorneal application of highly selective agonists for prostaglandin E2 receptor subtypes in pigmented rabbits: effect of tetramethylpyrazine.

We examined the disruptive effect of highly selective agonists for prostaglandin E2 receptor subtypes (EP1, EP2, EP3 and EP4) on the blood-aqueous barrier, and evaluated the inhibitory effect of tetramethylpyrazine, an active component of Ligusticum wallichii, on the elevation of aqueous flare induced by the EP agonists in pigmented rabbits. Highly selective EP agonists (ONO-DI-004, EP1 agonist; ONO-AE1-259-01, EP2 agonist; ONO-AE-248, EP3 agonist; ONO-AE1-329, EP4 agonist) at 12.5 to 250 microg/ml were transcorneally administered to the eyes of pigmented rabbits using a glass cylinder. Animals were pretreated intravenously with tetramethylpyrazine (10 or 30 mg/kg) 30 minutes before application of the EP2 or the EP4 agonist. Aqueous flare was measured using a laser flare-cell meter. Aqueous flare intensity was expressed as the area under the curve (AUC) in arbitrary units. After administration of ONO-AE1-259-01 or ONO-AE1-329, aqueous flare increased and then gradually decreased. ONO-DI-004 and ONO-AE-248 had almost no effect on aqueous flare elevation. The AUC of eyes in rabbits pretreated with tetramethylpyrazine, 10 or 30 mg/kg i.v., was significantly smaller than that of eyes in rabbits treated with ONO-AEI-259-01 alone. The AUC of eyes in rabbits pretreated with tetramethylpyrazine, 10 or 30 mg/kg i.v., was not significantly smaller than that of eyes in rabbits treated with ONO-AEI-329 only. The results indicated that EP2 and EP4 agonists induced aqueous flare elevation in pigmented rabbits, and that tetramethylpyrazine inhibited the aqueous flare elevation induced by the EP2 agonist but did not suppress the elevation induced by the EP4 agonist.     

Activation of Rice Aminopeptidase by Anions and Some Properties of the Enzyme

In order to determine which proteases are responsible for the autolysis of krill, the effects of several protease inhibitors on the autolysis and protease activities of krill were investigated.

Homogenates of whole bodies, and the cephalothorax and abdomen parts of frozen krill were equilibrated at 37°C at different pHs between 2 to 10 and allowed to stand for 16 hr, following which the increase in the TCA soluble fraction was monitored. ¹⁴C-Hemoglobin (¹⁴C-Hb) hydrolyzing activity was also measured using each homogenate as a crude enzyme preparation. The degree of autolysis and the ¹⁴C-Hb hydrolyzing activity were maximum at pH 5 ~ 8 for the parts studied. The hydrolytic activity was highest in the cephalothorax, followed by that in the whole body and then the abdomen.

The effects of inhibitors on the ¹⁴C-Hb hydrolyzing activity were examined, and it was seen that soybean trypsin inhibitor (STI), diisopropyl fluorophosphate (DFP) and leupeptin significantly inhibited the activity at neutral pH, and pepstatin, monoiodoacetic acid (IAAcid) and leupeptin were effective at acidic pH for all the parts. Investigation of the effects of inhibitors on the autolysis at 20°C at pH 4 and 7 by SDS–polyacrylamide gel electrophoresis indicated that the autolysis of the cephalothorax and whole body at pH 7 was suppressed a little by STI and the autolysis of the abdomen and whole body at pH 4 was significantly inhibited by iodoacetamide (IAA) and leupeptin.

These results suggest that the main proteases responsible for the autolysis of krill are trypsin like-proteases at neutral pH and cathepsins (B, H and L types) at acidic pH.