Synthesis of an Isoglutathione Derivative by Proteases and Its α→γ Transpeptidation

Benzyloxycarbonyl-l-cysteine and glycine benzhydrylamide were condensed by papain in a yield of 39.5%. After elimination of the N-protecting group, l-cysteinylglycine benzhydrylamide was condensed with benzyloxycarbonyl-l-glutamic acid by acid protease from Irpex lacteus Fr. in a yield of 21.0%. From the isoglutathione derivative thus obtained, a γ-linkage between glutamic acid and cysteine was formed by α → γ transpeptidation in alkaline conditions after esterification of γ-carboxylic acid.     

Ultrastructural study of plasmodesmata in the brown alga Dictyota dichotoma (Dictyotales, Phaeophyceae)

Plasmodesmata are intercellular bridges that directly connect the cytoplasm of neighboring cells and play a crucial role in cell-to-cell communication and cell development in multicellular plants. Although brown algae (Phaeophyceae, Heterokontophyta) are phylogenetically distant to land plants, they nevertheless possess a complex multicellular organization that includes plasmodesmata. In this study, the ultrastructure and formation of plasmodesmata in the brown alga Dictyota dichotoma were studied using transmission electron microscopy and electron tomography with rapid freezing and freeze substitution. D. dichotoma possesses plasma membrane-lined, simple plasmodesmata without internal endoplasmic reticulum (desmotubule). This structure differs from those in land plants. Plasmodesmata were clustered in regions with thin cell walls and formed pit fields. Fine proteinaceous "internal bridges" were observed in the cavity. Ultrastructural observations of cytokinesis in D. dichotoma showed that plasmodesmata formation began at an early stage of cell division with the formation of tubular pre-plasmodesmata within membranous sacs of the cytokinetic diaphragm. Clusters of pre-plasmodesmata formed the future pit field. As cytokinesis proceeded, electron-dense material extended from the outer surface of the mid region of the pre-plasmodesmata and finally formed the nascent cell wall. From these results, we suggest that pre-plasmodesmata are associated with cell wall development during cytokinesis in D. dichotoma.     

Photosynthetic activity of a temperate coral Acropora pruinosa (Scleractinia, Anthozoa) with symbiotic algae in Japan

Physiological properties of the temperate hermatypic coral Acropora pruinosa Brook with symbiotic algae (zooxanthellae) on the southern coast of the Izu Peninsula, Shizuoka Prefecture, central Japan, were compared between summer and winter. Photosynthesis and respiration rates of the coral with symbiotic zooxanthellae were measured in summer and winter under controlled temperatures and irradiances with a differential gasvolumeter (Productmeter). Net photosynthetic rate under all irradiances was higher in winter than in summer at the lower range of temperature (12–20°C), while lower than in summer at the higher range of temperature (20–30°C). The optimum temperature for net photosynthesis was apt to fall with the decrease of irradiance both in summer and winter, whereas it was higher in summer than in winter under each irradiance. At 25/ 50/100 μmol photons nr² s^?1, it was nearly the sea‐water temperature in each season. Dark respiration rate was higher in winter than in summer, especially in the range from 20–30°C. In both seasons the optimum temperature for gross photosynthesis was 28°C under 400 μmol photons nr² s^?1 and lowered with decreasing irradiance up to 22°C under 25 μmol photons nr² s^?1 in summer, while 20°C under the same irradiance in winter. The optimum temperature for production/respiration (P/R) ratio was higher in summer than in winter under each irradiance. Results indicated that metabolism of coral and zooxanthellae is adapted to ambient temperature condition under nearly natural irradiance in each season.     

Heat-treatment method for producing fatty acid-bound alpha-lactalbumin that induces tumor cell death

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), which was identified in human breast milk as an alpha-lactalbumin (LA)-oleic acid complex, kills tumor cells, selectively. Although it may have potential as a therapeutic agent against various tumor cells, only low-volume methods for its production exist. In this study, heat treatment was used to produce complexes from LAs and oleic acid using a simple method. In the case of human LA and oleic acid, heat-treated samples apparently showed much stronger activities than those treated at room temperature, with cytotoxicities equal to that of HAMLET. Furthermore, circular dichroism spectroscopy revealed that heat-treated samples lost their tertiary structure, suggesting a molten globule as oleic acid-bound LA. BLA samples also showed strong activities by heat treatment. Batch production with heat treatment can efficiently convert LAs into tumoricidal complexes.     

A novel beta-defensin structure: a potential strategy of big defensin for overcoming resistance by Gram-positive bacteria

Big defensin is a 79-residue peptide derived from hemocytes of the Japanese horseshoe crab. It has antimicrobial activities against Gram-positive and -negative bacteria. The amino acid sequence of big defensin can be divided into an N-terminal hydrophobic half and a C-terminal cationic half. Interestingly, the trypsin cleaves big defensin into two fragments, the N-terminal and C-terminal fragments, which are responsible for antimicrobial activity against Gram-positive and -negative bacteria, respectively. To explore the antimicrobial mechanism of big defensin, we determined the solution structure of mature big defensin and performed a titration experiment with DPC micelles. Big defensin has a novel defensin structure; the C-terminal domain adopts a beta-defensin structure, and the N-terminal domain forms a unique globular conformation. It is noteworthy that the hydrophobic N-terminal domain undergoes a conformational change in micelle solution, while the C-terminal domain remains unchanged. Here, we propose that the N-terminal domain achieves its antimicrobial activity in a novel fashion and explain that big defensin has developed a strategy different from those of other beta-defensins to suppress the growth of Gram-positive bacteria.     

Spontaneous asparaginyl deamidation of canine milk lysozyme under mild conditions

Asparaginyl deamidation is a common form of nonenzymatic degradation of proteins and peptides. As it introduces a negative charge spontaneously and irreversibly, charge heterogeneity can be accumulated in protein solution during purification, preservation, and experiments. In this study, canine milk lysozyme (CML), a useful model for the study of the molten globule state, exhibited charge heterogeneity after sample purification. Four Asn residues in CML deamidated rapidly under mild conditions: pH 8.0 and 30 degrees C. Other than these residues, one Asn residue, which was stable in the native state, was labile to deamidation in the unfolded state. This suggests that the structural formation around Asn can suppress deamidation. Substitutions of these labile Asn residues to Gln residues prevented deamidation effectively. Because the substitutions did not disrupt the structural formation of the native and molten globule states, they will enable more precise analyses for physical and structural studies.     

Morphometric study of Ecklonia cava (Laminariales, Phaeophyta) sporophytes in two localities with different temperature conditions

Sporophytes of Ecklonia cava Kjellman (Laminariales, Phaeophyta) were collected seasonally from within 3–4 replicate, 1‐m² quadrates, haphazardly placed in dense assemblages at 6–9 m depth in Tei, Tosa Bay (southern Japan; water temperature 15–29°C) from 1995 to 1996, and in Nabeta Bay, Shimoda (central Japan; water temperature 13–25°C) from 1996 to 1997. Growth rings were checked for all samples and mean values of each biometric parameter of sporophytes 1‐year‐old and over were compared. The plant length (stipe length + primary blade length) was always shorter in Tei sporophytes (24–52 cm) than Nabeta sporophytes (70–100 cm), the difference being mainly owing to the shorter stipe length in Tei sporophytes (7–14 cm) than in Nabeta sporophytes (54–83 cm). However, the primary blade length was sometimes longer in Tei sporophytes (12–38 cm) than in Nabeta sporophytes (14–21 cm). Stipe diameter, longest bladelet length and primary blade width were mostly less in Tei sporophytes (8.6–12.4 mm, 5.5–7.0 cm and 23.4–38.0 cm, respectively) than Nabeta sporophytes (16.4–20.2 mm, 9.2–12.0 cm and 43.0–52.6 cm, respectively). Nevertheless, the number of bladelets of sporophytes from Tei (15–28) and Nabeta (18–29) were within the same range. At Tei, 32–43% of the sporophytes had wrinkled blades in summer and autumn, whereas wrinkled primary blades or bladelets were not observed in Nabeta sporophytes throughout the experimental period. These morphometric differences of E. cava between the two localities are suggested to be dependent on environmental factors, especially seawater temperature.     

Effects of a helix substitution on the folding mechanism of bovine alpha-lactalbumin

The structure, stability, and unfolding-refolding kinetics of a chimeric protein, in which the amino acid sequence of the flexible loop region (residues 105-110) comes from equine lysozyme and the remainder of the sequence comes from bovine alpha-lactalbumin were studied by circular dichroism spectroscopy and stopped-flow measurements, and the results were compared with those of bovine alpha-lactalbumin. The substitution of the flexible loop in bovine alpha-lactalbumin with the helix D of equine lysozyme destabilizes the molten globule state, although the native state is significantly stabilized by substitution of the flexible loop region. The kinetic refolding and unfolding experiments showed that the chimeric protein refolds significantly faster and unfolds substantially slower than bovine alpha-lactalbumin. To characterize the transition state between the molten globule and the native states, we investigated the guanidine hydrochloride concentration dependence of the rate constants of refolding and unfolding. Despite the significant differences in the stabilities of both the molten globule and native states between the chimeric protein and bovine alpha-lactalbumin, the free energy level of the transition state is not affected by the amino acid substitution in the flexible loop region. Our results suggest that the destabilization in the molten globule state of the chimeric protein is caused by the disruption of the non-native interaction in the flexible loop region and that the disruption of the non-native interaction reduces the free energy barrier of refolding. We conclude that the non-native interaction in the molten globule state may act as a kinetic trap for the folding of alpha-lactalbumin.     

A practical method for measuring deoxynivalenol, nivalenol, and T-2 + HT-2 toxin in foods by an enzyme-linked immunosorbent assay using monoclonal antibodies

We have developed and tested an enzyme-linked immunosorbent assay system for individual measurement of deoxynivalenol, nivalenol, and T-2 + HT-2 toxin using monoclonal antibodies for 3,4,15-triacetyl-nivalenol, for both 3,4,15-triacetyl-nivalenol and 3,15-diacetyl-deoxynivalenol, and for acetyl-T-2 toxin. The assay system comprised three kits (desinated the DON + NIV kit, the NIV kit, and the T-2 + HT-2 kit). The practical performance of the enzyme-linked immunosorbent assay system was assessed by assaying trichothecene mycotoxins in wheat kernels. The enzyme-linked immunosorbent assay system meets all the requirements for use in a routine assay in terms of sensitivity (detection limit: deoxynivalenol 80 ng/g, nivalenol 80 ng/g, T-2 toxin 30 ng/g), reproducibility (total coefficient of variation: 1.9-6.2%), accuracy (recovery: 93.8-112.0%), simplicity and rapidity (time required: <2 h), mass handling (>42 samples/assay), and a good correlation with gas chromatography-mass spectrometry (r=0.9146-0.9991). Components derived from the wheat extract did not interfere with the assay kits. The enzyme-linked immunosorbent assay system is a useful alternative method to gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, or liquid chromatography-ultraviolet absorption for screening cereals and foods for trichothecene mycotoxin contamination.     

Endoplasmic reticulum quality control regulates the fate of transthyretin variants in the cell

The secretion of transthyretin (TTR) variants contributes to the pathogenesis of amyloidosis because they form aggregates in the extracellular environment. However, the mechanism of how TTR variants pass the quality control system in the endoplasmic reticulum (ER) has not yet been elucidated. We investigated here the mechanism of how TTR passes ER monitoring. Monomeric mutation introduced in TTRs (M-TTRs) resulted in the ER retention of amyloidogenic M-TTRs but not non-amyloidogenic M-TTRs. Retention of amyloidogenic M-TTRs induced the unfolded protein response and upregulated the expression of ER chaperones BiP and glucose-regulated protein (GRP) 94. Additionally, we showed that the ER-retained amyloidogenic M-TTRs are subject to ER-associated degradation. On the other hand, the amyloidogenic TTR variants and non-amyloidogenic M-TTRs were secreted normally. These findings suggest that unlike for wild-type TTR, the ER quality control system may differentially regulate the fate of the TTR variants and their monomeric counterparts.