Characterization of interleukin 2 stimulated 65-kilodalton phosphoprotein in human T cells

We have characterized the cellular proteins which are rapidly phosphorylated by interleukin 2 (IL 2) in a human IL 2 dependent cell line. When treated with IL 2, the phosphorylation of five proteins, 65, 50, 37, 24, and 21 kDa, was found in IL 2 dependent cell lines by two-dimensional gel electrophoretic analysis. After cell conversion from an IL 2 dependent state to an IL 2 independent state, one of the five phosphoproteins, the 65-kDa protein, became constitutively phosphorylated even without addition of IL 2. Also, in other IL 2 independent cell lines, such as KUT-2 and HUT-102, constitutive phosphorylation of the 65-kDa protein occurred without IL 2-stimulation. So our researchers were focused on biochemical characterization of the 65-kDa protein. It was found that the 65-kDa protein was one of the major cellular proteins by comparing the results of two-dimensional gel electrophoretic analysis of [32P]Pi-labeled and [3H]leucine-labeled cellular proteins and peptide mapping analysis. Subcellular fractionation studies indicated that the 65-kDa protein is a cytosol protein. The 65-kDa protein was purified from cytosol of a human T cell line, and its amino acid composition and amino acid sequences of its three oligopeptides were determined. It was found that the 65-kDa protein is identical with 1-plastin.     

Probable Contribution of Protein Phosphorylation by Protein Kinase C to Spicule Formation in Sea Urchin Embryos

The formation of spicules and development of pluteus arms in sea urchin embryos were strongly blocked by H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) but were not affected by HA1004 ( N -(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). Archenteron formation occurred normally in the presence of these compounds. Late gastrulae (28 hr after fertilization) were exposed to ³²Pi for 30 min at 20°C, and then dissociated and their primary mesenchyme cells with spicules, embryo-wall cells and archenteron cells were separated. Then, the radioactivities in the protein fractions of these separated cells were measured. Results showed that culture of embryos with H-7 strongly inhibited ³²p incorporation into proteins in primary mesenchyme cells but caused little inhibition of its incorporations in embryo-wall cells and archenteron cells. The effective concentrations of H-7 for inhibition of ³²p incorporation were within the range that blocked spicule formation and growth of pluteus arms in embryos. HA1004 only slightly inhibited ³²p incorporation into protein in mesenchyme cells, embryo-wall cells and archenteron cells of embryos exposed to ³²Pi. Protein kinase C activity was detectable only in isolated primary mesenchyme cells associated with spicule structures. These suggest that phosphorylation of proteins by protein kinase C contributes to the formation of spicule structures.     

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin: A spectrin-like calmodulin-binding protein (fodrin)

In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated M_r of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca²⁺ and calmodulin. Peak II revealed a basal activity in the absence of Ca²⁺ and calmodulin, which was stimulated 2-fold by addition of Ca²⁺. Calmodulin had no effect on the peak II activity.     

Two new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) and Bacillus thuringiensis subsp. tochigiensis (serotype 19)

Bacteriological and serological characteristics of three Bacillus thuringiensis isolates obtained in Japan were investigated. They formed typical rhomboidal parasporal inclusions but flagellar (H) antigens of these isolates were different from those of the known 17 H serotypes of B. thuringiensis. The three isolates were divided into two new serotypes (serotypes 18 and 19). The serotype 18 isolate (3–71) produced thermostable exotoxin and the inclusions of this isolate were toxic to larvae of the silkworm, Bombyx mori, but nontoxic to larvae of the mosquito, Aedes aegypti. The other isolate (119-72) belonging to serotype 18 produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. However, other bacteriological properties of the isolate 119-72 were similar to those of the isolate 3–71. The serotype 19 isolate (117-72) produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. Acid production from saccharose and the production of brownish purple pigment were observed in the two serotype 18 isolates, while neither of them was observed in the serotype 19 isolate. In other 29 biochemical properties tested, there was no difference among the three isolates. Based on these characteristics, the following two subspecies names are proposed: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) for the type strain 3–71 and Bacillus thuringiensis subsp. tochigiensis (serotype 19) for the type strain 117-72.     

Angiostrongylus cantonensis: Rejection of pulmonary arterial transfers of lung stage worms

Experimental transfer of the lung stage worms of Angiostrongylus cantonensis was performed between permissive hosts (rats) and between permissive (rat) and nonpermissive hosts (guinea pigs and rabbits). These worms from rats were rejected when implanted into nonpermissive hosts. Unexpectedly, similar worms did not survive well even in permissive hosts; the majority of recipient rats did not have first-stage larvae (L₁) in their stools and, even when positive for L₁, the number of the larvae shed was few. These findings contrast with the successful pulmonary arterial transfer of younger, intracranial-stage worms. It was shown that differences in rat strain between donor and recipient had no significant effect on the subsequent worm survival in recipient hosts. The alteration of maintaining conditions of the intrapulmonary worms, prior to transfer, in terms of temperature, media, and maintaining period, also showed no profound effect on the subsequent worm survival. The kinetics of precipitating and reaginic antibody levels in rats implanted with the intrapulmonary worms were analogous to those in rats with intracranial-stage worms. The findings indicate that some qualitative differences may exist between the worms obtained from two different sites.     

Limited autolysis of Ca2+-activated neutral protease (CANP) changes its sensitivity to Ca2+ ions

Ca2+-activated neutral protease (CANP) usually requires mM Ca2+ for activation. The sensitivity of CANP to Ca2+ is greatly enhanced by passing it through a casein-Sepharose column in the presence of Ca2+ ions. This conversion is ascribed to autolysis of CANP. The converted enzyme required 40 microM Ca2+ for 50% activation. Various properties of the converted enzyme were very similar to those of CANP-I, recently found in canine heart muscle. Names of "m-CANP" and "mu-CANP" are proposed for CANPs which require mM and microM order Ca2+ for inactivation, respectively.     

One step purification procedure of elastase from pancreatic powder by affinity chromatography

Pancreatic elastase was isolated from acetone powder of porcine pancreas by a one step purification procedure on a trialanyl CH-Sepharose 4B affinity column. This column exhibited affinity not only for active elastase but also for trypsin and chymotrypsin which were present in the same pancreatic powder. However, as the extent of affinity toward elastase is considerably higher, the proper conditions were determined with which the adsorbed elastase was isolated in a highly purified form. The yield of elastolytic activity ranged from 60–85% and the purified elastase was shown to be one component by polyacrylamide disc electrophoresis.     

In vitro construction of bacteriophage λ and plasmid DNA molecules containing DNA fragments from bacteriophage T4

Restriction endonucleases EcoRI and HindIII generated fragments of T4 cytosine-containing DNA were inserted into bacteriophage vector λgtSu_III and plasmid vectors pMB9 and pBR313. Resulting clones were screened for hybridization with ³²P labeled T4 tRNA. Recombinant bacteriophages and plasmids were isolated which contained a T4 fragment coding for T4 RNA species 1 and 2 and T4 tRNA^Arg. Selected λ-T4 hybrid bacteriophages were grown to high titer and their DNA analyzed by gel electrophoresis.