Citrullinated inter-alpha-trypsin inhibitor heavy chain 4 in arthritic joints and its potential effect in the neutrophil migration.

The citrullinated inter-alpha-trypsin inhibitor heavy chain 4 (cit-ITIH4) was identified as its blood level was associated with the arthritis score in peptide glucose-6-phosphate-isomerase-induced arthritis (pGIA) mice and the disease activity in patients with rheumatoid arthritis (RA). This study aimed to clarify its citrullination pathway and function as related to neutrophils. In pGIA-afflicted joints, ITIH4 and cit-ITIH4 levels were examined by immunohistochemistry (IHC), immunoprecipitation (IP) and Western blotting (WB), while peptidylarginine deiminase (PAD) expression was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), IHC and immunofluorescent methods. The pGIA mice received anti-lymphocyte antigen 6 complex locus G6D (Ly6G) antibodies to deplete neutrophils and the expression of cit-ITIH4 was investigated by WB. The amounts of ITIH4 and cit-ITIH4 in synovial fluid (SF) from RA and osteoarthritis (OA) patients were examined by I.P. and W.B. Recombinant ITIH4 and cit-ITIH4 were incubated with sera from healthy volunteers before its chemotactic ability and C5a level were evaluated using Boyden's chamber assay and enzyme-linked immunosorbent assay (ELISA). During peak arthritic phase, ITIH4 and cit-ITIH4 were increased in joints while PAD4 was over-expressed, especially in the infiltrating neutrophils of pGIA mice. Levels of cit-ITIH4 in plasma and joints significantly decreased upon neutrophil depletion. ITIH4 was specifically citrullinated in SF from RA patients compared with OA patients. Native ITIH4 inhibited neutrophilic migration and decreased C5a levels, while cit-ITIH4 increased its migration and C5a levels significantly. Cit-ITIH4 is generated mainly in inflamed joints by neutrophils via PAD4. Citrullination of ITIH4 may change its function to up-regulate neutrophilic migration by activating the complement cascade, exacerbating arthritis.

Void structure in colloidal dispersions.

The time evolution of void structures in highly purified polymer latex dispersions was studied with a confocal laser scanning microscope. In such dispersions, which were initially homogeneous, the voids grew with time when the dispersions were kept standing and formed more quickly in the internal material than in material close to the glass-dispersion interface. Void formation is thus not an artifact arising from the presence of the interface. A similar structural inhomogeneity, in apparently homogeneous systems, is discussed for simple ionic solutions, ionic polymer solutions, and Langmuir-Blodgett films.

Pressure dependence of trypsin-catalyzed hydrolyses of specific substrates.

The effects of pressure on the trypsin-catalyzed hydrolyzed hydrolyses of three specific substrates, N-benzoyl-L-arginine ethyl ester (BzArgOEt), amide (BzArgNH2) and p-nitroanilide (BzArgNA), have been examined. The volume of the activation (delta V++) for kcat was -2.4 ml/mol for BzArgOEt and +3 - +6 ml/mol for BzArgNH2. Because of different rate-determining steps in the steady-state kinetics, the delta V++ value for BzArgOEt would indicate the activation volume of the deacylation step, whereas that for BzArgNH2 the delta V++ for the acylation step. The activation volumes were accounted for in terms of the difference in the mechanisms on the formation and decomposition of the tetrahedral-like intermediates during the acylation and deacylation steps. The delta V values for the formation of BzArgNH2- and thionine-trypsin complexes were several ml/mol, consistent with the fact that the main driving force of the substrate binding to this enzyme is electrostatic interaction, and in contrast to the delta V values of alpha-chymotrypsin complex formation with indole (approximately 0 ml/mol) or 2-furylacryloyl-D-tryptophan methyl ester (approximately 0 ml/mol), for which the hydrophobic interaction is the dominant force of the substrate binding. For the hydrolysis of BzArgNA, which showed a distinct substrate activation at high substrate concentrations, the pressure dependence of the four parameters, ks, Ks, (the catalytic rate and dissociation constant of the normal enzyme-substrate complex, respectively), Kss and Kss (those of the complex activated by the binding of the second substrate molecule), were measured at 1 atm and 1000 atm (25 degrees C). All of the four parameters increased with increase in pressure.

Mutual recognition between polymerized liposomes: enzyme and enzyme inhibitor system.

In order to examine the usefulness of polymerized liposomes as a model for cell membranes, a mutual recognition phenomenon between different liposomes on which complementary ligands were attached was examined. We used trypsin- and soybean trypsin inhibitor (STI)-carrying polymerized liposomes to attain high sensitivities. The STI which was immobilized on the polymerized mono-dienoylphosphatidylcholine liposome showed a definite inhibitory effect on the catalytic activity of the trypsin which was immobilized on another polymerized liposome, whereas the inhibitory effect of the STI which was immobilized on the di-dienoylphosphatidylcholine liposome was much smaller than that of the mono-dienoylphosphatidylcholine system because of the larger rigity of the di-dienoylphosphatidylcholine liposome. These results suggest that the mutual recognition between complementary ligands can be realized by using polymerized liposomes with a physical stability and moderate deformability as their carriers.

pH dependence of the formation of dimeric alpha-chymotrypsin and its catalytic activity.

The dimerization of alpha-chymotrypsin (alpha-CT) has been known to involve a specific interaction between the Tyr-146 alpha-carboxyl of one molecule and the His-57 imidazole, which is a member of the catalytic triad in the active site, of the other. This interaction determines the pH dependences of the dimerization constant and of the catalytic activities of the monomeric and dimeric enzymes. We compared the pKa values for catalytic activities with known pKa values for the dimerization constant in order to assign pKa values to residues of the enzyme. In the monomeric enzyme, the catalytic triad has a pKa value of 3.6 at the site between the Asp-102 carboxyl and His-57 N delta 1, and the Tyr-146 alpha-carboxyl has a pKa value of 4.6. In the dimeric enzyme, the site between the Asp-102 carboxyl and His-57 N delta 1 has a pKa value of around 5.5 and the site between His-57 N epsilon 2 and the Tyr-146 alpha-carboxyl has a pKa value around 2.4. Protonation at the site between the Asp-102 carboxyl and His-57 N delta 1 reduced the catalytic activity of the dimeric enzyme for p-nitrophenyl propionate, indicating that the Asp-102 carboxyl plays an important role in the monomeric alpha-CT-catalyzed reactions.

Specific and non-specific effects of inert anions and cations on the dimerization of alpha-chymotrypsin and the catalytic activities of monomeric and dimeric alpha-chymotrypsins.

The effect of salts on the dimerization and the catalytic activity of dimeric alpha-chymotrypsin (alpha-CT) was investigated. The observed effect was mainly related to the anionic constituent of the salt, and its order depended on the salt concentration. The order of effect of anions on several parameters at [salt] less than 0.02 M was SO4 2-, ClO4-, NO3-, Br-, Cl-, which reflected the electrostatic interaction between the anion and the positively charged surface of alpha-CT. On the other hand, the anion dependence at moderate salt concentrations (0.1-0.2 M) followed the Hofmeister series, SO4 2-, Cl-, Br-, NO3-, ClO4-, which was related to the direct and indirect interactions between the anion and non-charged groups of the protein. The anion order for the dimerization constant of this enzyme, however, was the complete reverse of that generally observed in the aggregation of proteins at high salt concentration (greater than 1 M). This result was accountable for in terms of a specific interaction in the formation of the dimeric enzyme (probably that between the active site of one monomer and Tyr-146 of the other).

Interaction of specific amino acid derivatives with dimeric alpha-chymotrypsin.

Binding and catalytic activities of dimeric alpha-chymotrypsin from specific amino acid derivatives were investigated with special reference to the equilibrium between active and inactive monomeric forms of this enzyme occurring in a low pH range. The low catalytic activity of the dimeric enzyme towards these specific substrates was revealed to be due to the difficulty in binding of the dimer. However the free tryptophanates and N-acetyl-L-tryptophan, which are slightly smaller (in molecular size) than the above substrates and comparable to the nonspecific phenyl acetate substrates (towards which the dimeric alpha-chymotrypsin showed an distinct catalytic activity in our previous study [J. Biochem. 87, 871-880 (1980)]), were bound to the dimer more strongly than to the monomeric enzyme. Hence they enhanced dimer formation when added at low concentrations.

"Ordered" structure in ionic dilute solutions: dendrimers with univalent and bivalent counterions.

As an intermediate sample of ionic solutes between colloidal particles (macroions) and simple electrolyte ions, we made small-angle x-ray scattering (SAXS) measurements for aqueous solutions of poly(amido amine) dendrimers of three generations (G4, G7, and G10). The SAXS curves of univalent acid solutions showed a single scattering peak, as observed for synthetic macroions. The peak position was dependent on the dendrimer concentration but independent not only of the acid concentration (degree of protonation) but also of the counterion species. The effective charge density of the dendrimer determined by conductivity measurements was found to be insensitive to the acid concentration and the counterion species. The nearest neighbor interparticle distance 2D(exp) calculated from the peak position of the structure factor of G7 and G10 was obviously smaller, though slightly, than the average interparticle distance 2D(0) calculated from molecular weights and concentrations of dendrimers, implying that acid solutions of dendrimers formed the two-state structures by the attractive force. The ultra-small-angle x-ray scattering curve for the hydrochloric acid solution did not show an upturn, which indicates the existence of large scale structural inhomogeneities such as localized ordered structures, probably due to the weak attraction and hence less clear distinction of the ordered and disordered regions. For sulfuric acid solutions, clear scattering peaks were not observed. The bivalent counterions were more strongly associated with the dendrimer ions than the univalent ones. The resulting low charge number of the dendrimers with the bivalent counterion was confirmed directly by the conductivity measurements. These observations confirm that the counterion-mediated attraction does exist even with the univalent counterions and point out that the frequently advanced claim that the effective potential is essentially repulsive with univalent counterions while attraction appears with bivalent counterions is not necessarily correct. It is noted that the intensity of the counterion-mediated attraction in dendrimer solutions is dictated by both the effective charge density and the effective charge number, in contrast with macroionic solutions or colloidal dispersions in which only the effective charge density appeared to be important.

[Metastasis of uterine cervical cancer to the biceps muscle of right upper arm; report of a case].

A hematogenic metastasis to the biceps muscle of right upper arm was reported in a 48-year-old women with recurrent uterine cervical cancer who had undergone radiotherapy. Cancer metastasis to the limb skeletal muscle is very rare. We have not known the similar case report in uterine cervical cancer.