Summary: A major innate defense system in invertebrates is the melanization of pathogens and damaged tissues. This important process is controlled by the enzyme phenoloxidase (PO) that in turn is regulated in a highly elaborate manner for avoiding unnecessary production of highly toxic and reactive compounds. Recent progress, especially in arthropods, in the elucidation of mechanisms controlling the activation of zymogenic proPO into active PO by a cascade of serine proteinases and other factors is reviewed. The proPO-activating system (proPO system) is triggered by the presence of minute amounts of compounds of microbial origins, such as β-1,3-glucans, lipopolysaccharides, and peptidoglycans, which ensures that the system will become active in the presence of potential pathogens. The presence of specific proteinase inhibitors prevents superfluous activation. Concomitant with proPO activation, many other immune reactions will be produced, such as the generation of factors with anti-microbial, cytotoxic, opsonic, or encapsulation-promoting activities.

The prophenoloxidase-activating system in invertebrates.

Cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization.

Early events in crustacean innate immunity.

The cystine knot motif in toxins and implications for drug design.

https://www.cell.com/trends/biochemical-sciences/pdf/S0968-0004(00)01602-9.pdf

Tyrosinase/catecholoxidase activity of hemocyanins: structural basis and molecular mechanism.

The chemical synthesis of human interleukin-2 (IL-2) , having a core 1 sugar, by a ligation method is reported. Although IL-2 is a globular glycoprotein, its C-terminal region, in particular (99-133), is extremely insoluble when synthesized by solid-phase method. To overcome this problem, the side-chain carboxylic acid of the Glu residues was protected by a picolyl ester, thus reversing its polarity from negative to positive. This reverse polarity protection significantly increased the isoelectric point of the peptide segment and made it positive under acidic conditions and facilitated the purification. An efficient method to prepare the prolyl peptide thioester required for the synthesis of the (28-65) segment was also developed. These efforts resulted in the total synthesis of the glycosylated IL-2 having full biological activity.

Chemical Synthesis of O‐Glycosylated Human Interleukin‐2 by the Reverse Polarity Protection Strategy

Phenoloxidase inhibitor (POI), found in the hemolymph of housefly pupae, is a novel dopa-containing and cystine-rich peptide that competitively inhibits phenoloxidase with a Ki in the nanomolar range. [Tyr32]POI is a potential precursor molecule also found in the hemolymph that may be posttranslationally oxidized to the dopa-containing peptide after creation of a rigid structure. By employing both a solid-phase peptide synthesis system based on a 9-fluorenylmethoxycarbonyl strategy and a specific air oxidation technique to ensure correct folding, we have been able to synthesize [Tyr32]POI. The synthetic [Tyr32]POI was confirmed to be identical to the native [Tyr32]POI by coelution high-performance liquid chromatography analysis and by enzymatic analysis using the phenoloxidase inhibition assay. To determine the disulfide pairings within the peptides, a series of enzyme hydrolyses and partial reduction/alkylation steps were performed. Three cystine pairs (Cys11−Cys25, Cys18−Cys29, and Cys24−Cys36) were det...

A novel endogenous inhibitor of phenoloxidase from Musca domestica has a cystine motif commonly found in snail and spider toxins.

A heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli consists of 19 amino residues including 6 half-cystine residues. Analogs of STh from positions 6 to 19 with replacements of the Ala residue at position 14 by other amino acid residues were synthesized by a solid-phase method and examined by biological and biochemical methods. This Ala residue was demonstrated to be very important for expression of the toxicity of STh and for interaction of the toxin with its receptor protein(s).

Structure-activity relationship of Escherichia coli heat-stable enterotoxin: Role of Ala residue at position 14 in toxin-receptor interaction.

A method for the affinity capture of specific proteins from a complex mixture using a polyacrylamide gel technique is described. The approach is based on the orthogonal electro-transfer of proteins separated by ordinary polyacrylamide gel electrophoresis (PAGE) to a ligand-coupled polyacrylamide gel (Li-PAG), which is placed under the PAGE gel. Upon electro-transfer, the proteins orthogonally migrate from the PAGE into the Li-PAG, based on the net charge. During migration to the Li-PAG, proteins that specifically interact with a ligand can be transiently trapped in the Li-PAG, while those that do not interact with a ligand pass through it. This method permits the separation of the proteins that can specifically interact with a ligand, even when present in a complex mixture. The method is demonstrated by applying it to the one-step isolation of a trypsin inhibitor from a crude extract of soybean flour.

Affinity-trap polyacrylamide gel electrophoresis: a novel method of capturing specific proteins by electro-transfer.

PURPOSE:To manufacture a lumpy body of large-sized and solid wax, by a method wherein a wax compound obtained by compounding high-melting point wax to low-melting point wax is cast into a mold by performing blend treatment and cooling down to the vicinity of the melting temperature of a wax composition and solidified by leaving the same under a room temperature as it is. CONSTITUTION:A wax compound which is mainly comprised of low-melting point wax and obtained by compounding high-melting point wax to the same is blend-treated at 140-230 deg.C for 30-90min, in a manufacturing method of a lumpy body of large-sized wax. A molten matter of wax is cast into a mold after the same has been cooled down to the vicinity of its melting temperature while agitating under depressurization and solidified by leaving the same under a room temperature as it is. There is, for example, paraffin wax (melting point is 53 deg.C) as the low-melting point wax and polyethylene wax (melting point is 86 deg.C) and carnauba wax (melting point is 83 deg.C) and their compound are available as the high-melting point wax. A part of the same can be replaced with microcrystalline wax (melting point is 84 deg.C) to bring on microcrystallization.

Takashi Sato

Papers

Chemical Synthesis of O‐Glycosylated Human Interleukin‐2 by the Reverse Polarity Protection Strategy

A novel endogenous inhibitor of phenoloxidase from Musca domestica has a cystine motif commonly found in snail and spider toxins.

Structure-activity relationship of Escherichia coli heat-stable enterotoxin: Role of Ala residue at position 14 in toxin-receptor interaction.

Affinity-trap polyacrylamide gel electrophoresis: a novel method of capturing specific proteins by electro-transfer.

Manufacture of lumpy body of large-sized wax