Rabbit autoantibodies to actin induced by immunization with modified homologous actins

Actin is a major antigen involved in the reaction of smooth muscle antibody positive sera from patients with chronic active hepatitis. In the present study, actin extracted from rabbit skeletal muscle was denatured by sodium dodecyl sulfate and was immunized into the rabbit, a homologous animal for actin. The rabbits, thus immunized, produced antibodies reactive with actins of homologous and heterologous animals. In addition, the antibodies showed reactivity with autologous actin. It indicates that the denatured homologous actin is capable of terminating immunological tolerance to actin and induces formation of autoantibody to rabbit actin. This phenomenon may be implicated in the occurrence of anti-actin antibody in sera from patients with chronic liver disease and several other diseases.     

Chemotactic macrophage subpopulations defined by macrophage chemotactic factors from delayed hypersensitivity reaction sites

The chemotactic specificity of ia-positive and -negative macrophages was studied by using three macrophage chemotactic factors (MCF), -a, -b, and -c, isolated from delayed hypersensitivity reaction (DHR) skin sites in guinea pigs. Listeria-elicited macrophages migrated toward MCF-a, -b, and -c. The chemotactic responses suggested responsive subpopulations to MCF. The electronic programmable individual cell sorter (EPICS) was used to separate macrophages with anti-la monoclonal antibodies. Ia-positive subpopulations responded to MCF-c, although they did not migrate toward MCF-a and -b. In contrast, Ia-negative subpopulations migrated toward MCF-a and -b, but not toward MCF-c. Furthermore, MCF-c attracted Ia-positive macrophages, whereas MCF-a and -b were Ia-negative in vitro; MCF did not induce Ia-negative macrophages to express surface Ia-antigens in vitro. MCF-c was able to produce massive Ia-positive macrophage accumulations when injected i.p., whereas MCF-a accumulated Ia-negative macrophages. The data suggest that MCF-a and -b, which mediate initial macrophage reactions, attract Ia-negative macrophages, and that MCF-c, which mediates predominant macrophage reactions, attract Ia-positive macrophages in the DHR.     

Chemical, physicochemical and spectrophotometric properties of crystalline chlorophyll-protein complexes from Lepidium virginicum L

Two kinds of water-soluble chlorophyll-protein complexes were prepared from leaves of Lepidium virginicum L., one (CP661) from the plant cultivated in a green house from seeds collected near Mono Lake, CA, and the other (CP-663) from a plant collected at Narashino, Chiba, Japan, by ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose and Sephacryl S-200. The chlorophyll . proteins were further purified by crystallization. CP661 has absorption peaks at 661, 468, 439, 419, 380, 339 and 272 nm. CP663 had absorption peaks at 663, 469, 438, 419, 379, 338 and 272 nm. Estimated molecular weights were 78 000 for CP661 and 80 000 for CP663 by gel filtration chromatography and 83 000 for CP661 and 107 000 for CP663 by an equilibrium sedimentation method. 1 mol chlorophyll . protein contained 4 mol chlorophyll a and b with ratios of 1.0 in CP661 and 1.6 to 1.9 in CP663, but no carotenoids. These characters are different from those of chlorophyll-protein complexes which are prepared from the thylakoid membranes of chloroplasts with detergents.     

A 5-bp Insertion in Mip Causes Recessive Congenital Cataract in KFRS4/Kyo Rats

We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.     

Neoplastic cell transformation by heavy ions

We have studied the induction of morphological transformation by heavy ions. Golden hamster embryo cells were irradiated with 95 MeV 14N ions (530 keV/microns), 22 MeV 4He ions (36 keV/microns), and 22 MeV 4He ions with a 100-microns Al absorber (77 keV/microns) which were generated by a cyclotron at the Institute of Physical and Chemical Research in Japan. Colonies were considered to contain neoplastically transformed cells when the cells were densely stacked and made a crisscross pattern. It was shown that the induction of transformation was much more effective with 14N and 4He ions than with gamma or X rays. The relative biological effectiveness (RBE) relative to 60Co gamma rays was 3.3 for 14N ions, 2.4 for 4He ions, and 3.3 for 4He ions with a 100-microns Al absorber. The relationship between RBE and linear energy transfer was qualitatively similar for both cell death and transformation.     

On the anatomical definition of arterial networks in blood flow simulations: comparison of detailed and simplified models

Biomechanics and Modeling in Mechanobiology - The goal of this work is to assess the impact of vascular anatomy definition degree in the predictions of blood flow models of the arterial network. To...     

Precise identification of gene products in hearts after in vivo gene transfection, using Sendai virus-coated proteoliposomes.

Both efficient gene transfer and the exact identification of gene product are required for gene therapy. Gene transfection of green fluorescence protein (GFP) might be useful for the reporter. After in vivo cotransfection of GFP and beta-galactosidase (beta-Gal) genes in Sendai virus-coated proteoliposomes to rat hearts, we compared the sensitivity and specificity of three methods: GFP detection, histochemical staining (HC) of beta-Gal activity, and immunostaining (IS) of the beta-Gal protein. Fluorescence microscopy and double staining of HC and IS revealed that both GFP and IS were equally sensitive and fourfold superior to HC at the peak of gene expression. However, different from skeletal muscle, the GFP of transfected cardiomyocytes showed two demerits: the fluorescence quenching due to the intense staining of beta-Gal activity, and nonspecific autofluorescence from myocardium. Thus, specific IS would be so far the most reliable to identify the gene product in heart.     

Glycolytic activity of rat aorta after exercise

Activities of glycolytic enzymes in the aorta were investigated in female Wistar rats. There were two groups of rats; one served as the control (sedentary rats), while the other group was forced to run on a treadmill for 10 weeks. In the control animals, the activities of hexokinase, phosphofructokinase and aldolase were relatively lower than those of the other glycolytic enzymes (phosphoglucose isomerase, lactate dehydrogenase and pyruvate kinase). After exercise, the activity of phosphofructokinase increased by 15%, whereas the other enzymatic activities were much the same as in the controls. Within the limits of the experiments, the increased percentage of phosphofructokinase was statistically significant (p less than 0.05). Since phosphofructokinase is a putative rate limiting enzyme, this enzymatic activation may indicate that glycolytic activity in the rat aorta is enhanced during and after running exercise.     

Expression of bovine lung prostaglandin F synthase in Escherichia coli

The full-length bovine lung prostaglandin(PG) F synthase cDNA was constructed from partial cDNA clones and ligated into bacterial expression vector pUC8 to develop expression plasmid pUCPF1. This plasmid permitted the synthesis of bovine lung PGF synthase in Escherichia coli. The recombinant bacteria overproduced a 36-KDa protein that was recognized by anti-PGF synthase antibody, and the expressed protein was purified to apparent homogeneity. The expressed protein reduced not only carbonyl compounds including PGD2 and phenanthrenequinone but also PGH2; and the Km values for phenanthrenequinone, PGD2, and PGH2 of the expressed protein were 0.1, 100, and 8 microM, respectively, which are the same as those of the bovine lung PGF synthase. The protein produced PGF2 alpha from PGH2, and 9 alpha, 11 beta-PGF2 from PGD2 at different active sites. Moreover, the structure of the purified protein from Escherichia coli was essentially identical to that of the native enzyme in terms of C-terminal sequence, sulfhydryl groups, and CD spectra except that the nine amino acids provided by the lac Z' gene of the vector were fused to the N-terminus. These results indicate that the expressed protein is essentially identical to bovine lung PGF synthase. We confirmed that PGF synthase is a dual function enzyme catalyzing the reduction of PGH2 and PGD2 on a single enzyme and that it has one binding site for NADPH.