Macrophage chemotactic factor (MCF) produced by a human T cell hybridoma clone

A human T cell hybridoma clone, D6-18, producing high levels of macrophage chemotactic factor (MCF) was established by the emetine-actinomycin D selection method. MCF was found to be present not only in the culture medium but also in the cell lysate of D6-18 cells. The secretion of the MCF from D6-18 cells was effectively inhibited by disodium cromoglycate, which is an inhibitor of the degranulation of mast cells, suggesting that MCF is stored in granules. The MCF of D6-18 cells was purified from the sonicated cell lysate by ion-exchange chromatographies and high-performance liquid chromatography. The amino acid sequence of the purified MCF was revealed to be WLGREDGSE or WLGRQDGSE. The synthetic peptide WLGREDGSE showed chemotactic activity against guinea pig macrophages and human monocytes at the concentration of about 10(-8) M.     

Nitrilase of Rhodococcus rhodochrous J1. Purification and characterization

Nitrilase was purified from an extract of isovaleronitrile-induced cells of Rhodococcus rhodochrous J1 in seven steps. In the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous by polyacrylamide electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 78 kDa and consists of two subunits identical in molecular mass. The purified enzyme exhibits a pH optimum of 7.6 and a temperature optimum of 45 degrees C. The enzyme catalyzed stoichiometrically the hydrolysis of benzonitrile to benzoic acid and ammonia, and no formation of amide was detected. The enzyme required thiol compounds such as dithiothreitol, L-cysteine or reduced glutathione to exhibit maximum activity. The enzyme was specific for nitrile groups attached to an aromatic or heteroaromatic ring, e.g. benzonitrile, 3-chlorobenzonitrile, 4-tolunitrile, 2-furonitrile and 2-thiophenecarbonitrile. The comparison of the properties of the enzyme with other nitrilases and nitrile hydratases has been also discussed.     

Purification of GTP-binding proteins from bovine brain membranes. Identification of heterogeneity of the alpha-subunit of Go proteins

Using high-resolution Mono Q column chromatography, we purified 6 distinct peaks of GTP-binding proteins from bovine brain membranes. Five of them consisted of 3 polypeptides with alpha beta gamma-subunits and served as the substrate of islet-activating protein (IAP), pertussis toxin. The other one was purified as alpha-subunit alone and was also ADP-ribosylated by IAP in the presence of beta gamma-subunits. When each alpha-subunit was characterized by immunoblot analysis using various antibodies with defined specificity, the two of them were identified as Gi-1 and Gi-2, and other 4 appeared to be Go or Go-like G proteins. The alpha-subunits of immunologically Go-like proteins were apparently distinguishable from one another on elution profiles from the Mono Q column. Thus, there was a heterogeneity of the alpha-subunit of Go in the brain membranes.     

Nippostrongylus brasiliensis: radioresistant IgE antibody-forming cells in infected rats

In Nippostrongylus brasiliensis-infected rats, anti-N. brasiliensis IgE antibody production was observed at 20 weeks postinfection, long after the worms, as a source of antigen, had been expelled. The persistent IgE production was not abrogated after whole body irradiation (800 R) administered at 12 or 20 weeks, suggesting the participation of radioresistant IgE-forming cells. Help of T cells and recruitment of B memory cells in the irradiated rats seems to be ruled out by the findings that the irradiation completely inhibited the initiation of anti-N. brasiliensis IgE production in rats shortly after the infection with N. brasiliensis or after primary and secondary immunization with N. brasiliensis-antigen. Moreover, clearance of anti-N. brasiliensis IgE antibody from circulation did not seem to be crucially affected by the irradiation. The radioresistant cells forming anti-N. brasiliensis IgE were most productive in mesenteric lymph nodes as compared to other lymph nodes. The recognition of antigens fractionated by chromatography on Sephadex G-200 was the same for IgE-forming cells from rats 12 weeks after infection as for those from 3 weeks after infection. Based on these results, one of the mechanisms of persistent elevation of IgE antibody in the host infected with helminth parasites might be explained by the participation of radioresistant IgE-forming cells.     

Dual loading of the fluorescent indicator fura-2 and 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) in isolated myocytes

Isolated rat heart myocytes were loaded with both the Ca2+ sensitive fluorescent probe fura-2/AM and the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM). Changes in [Ca2+]i and pHi were measured simultaneously using digitized video fluorescence microscopy. In measurement of [Ca2+]i and pHi, the ratios of dual-loaded cells were not different from single-loaded cells. Using this method, [Ca2+]i and pHi in myocytes were 48 +/- 7 nM and 7.17 +/- 0.05. It is concluded that [Ca2+]i and pHi could be measured simultaneously in isolated myocyte using dual-loading of fura-2 and BCECF.     

Functions of the COOH-terminal region of cyclodextrin glucanotransferase of alkalophilic Bacillus sp. #1011: relation to catalyzing activity and pH stability

Cyclodextrin glucanotransferase (beta-CGTase) of alkalophilic Bacillus sp. #1011 degrades starch to mainly beta-cyclodextrin (beta-CD). This enzyme is considered to contain an extra-polypeptide in its COOH-terminal region in addition to its NH2-terminal domain which exhibits the starch-degrading activity. To analyze the functions of this extra-polypeptide in the beta-CGTase, two mutated enzymes, in which DNA regions encoding 10 or 13 amino acids from the COOH-terminus were deleted, were obtained. The mutated enzymes degraded starch to glucose, maltooligosaccharides and alpha-CD, in addition to beta-CD. Furthermore, the pH stability of the mutated enzymes in the alkaline pH range (pH 9-11) was reduced.     

Effect of pH on giant cell formation induced by human immunodeficiency virus ( HIV )

The rate of multinucleated giant cell formation by the fusion of HIV chronically infected human T-cells (MOLT-4/HIV) and HIV uninfected MOLT-4 cells was examined under various pH conditions. The number of giant cells formed under the different pH conditions was quantitatively monitored by "MULTISIZER". The rate of giant cell formation was significantly less at the pH lower than 6 but not influenced at higher pH. Plaque assay under various pH conditions revealed that inhibition of giant-cell formation at lower pH was not due to the influence over the recognition between gp120 of HIV and CD4 molecules.     

A case of sporotrichosis caused by two genetically differentSporothrix schenckii strains

A survey for the natural occurrence of Fusarium mycotoxins, deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN), in Dutch cereals (totaling 29 samples) harvested in 1984/1985, showed that 90%, 79% and 62% of samples were contaminated with DON, NIV and ZEN, respectively. Average contents (ng/g) in the total of positive samples were 221 (DON), 123 (NIV) and 61 (ZEN). Among the cereals examined, the highest concentrations (ng/g) was 3198 (DON), 1875 (NIV) and 677 (ZEN) in a yellow corn sample for animal feed. The results of this survey show that Dutch cereals were relatively significantly contaminated with Fusarium mycotoxins.     

The 76 kD cell-adhesion factor from crayfish haemocytes promotes encapsulation in vitro

Summary Semigranular cells from the crayfish, Pacifastacus leniusculus, were separated by Percoll gradient centrifugation and were used to study the encapsulation of foreign particles. The semigranular cells were found strongly to encapsulate glass beads coated with haemocyte lysate in which the prophenoloxidase-activating system had been activated with laminarin or with a low concentration of calcium ions. The granular cells only weakly encapsulated these particles. The encapsulationpromoting factor was purified from haemocyte lysates and found to be a 76 kD protein which was recognized by an antiserum to the previously described 76 kD cell-adhesion factor. After the last step in purification (Con A-Sepharose chromatography), the flowthrough consisted of several proteins, which had some, but less, encapsulation-promoting activity and contained a 30 kD band that was also recognized by the antiserum to the 76 kD cell-adhesion factor. If the haemocyte lysate prepared in low [Ca²⁺] was incubated with a -1,3-glucan prior to purification, no 76 kD protein could be isolated but only a 30 kD protein. The 30 kD protein thus seems to be a degradation product of the 76 kD cell-adhesion factor. We conclude that the 76 kD protein which is released from degranulating haemocytes, and to a lesser extent its 30 kD fragment, can promote encapsulation. Phenoloxidase did not have any encapsulation-promoting activity.     

Microfibrils: a constitutive component of reticular fibers in the mouse lymph node

Summary Fibrous components other than collagen fibrils in the reticular fiber of mouse lymph node were studied by electron microscopy. Bundles of microfibrils not associated by elastin and single microfibrils dispersed among collagen fibrils were present. The diameter of the microfibrils was 13.29±2.43 nm (n=100). Elastin-associated microfibrils occurred at the periphery of the reticular fiber. Elastin was enclosed by microfibrils, thus forming the elastic fiber, which was clearly demonstrated by tannic acid-uranyl acetate staining. In the reticular fiber of lymph nodes, the elastic fiber consisted of many more microfibrils and a small amount of elastin. These microfibrils, together with the collagen fibrils, may contribut to the various functions of the reticular fibers.