Membrane-bound lipoxygenase of rat cerebral microvessels

The microvessels isolated from rat cerebral cortex has arachidonate lipoxygenase activity, which was not due to possible contamination of the platelets. The major product was identified to be 12-hydroxyeicosatetraenoic acid. After homogenization and sonication of the microvessel preparations, the lipoxygenase activity was recovered both in the membrane- and the cytosol-fractions, whereas that in the platelets was recovered in the cytosol fraction. Membrane-bound lipoxygenase of the microvessels has apparent Km value of 3.8 microM for arachidonic acid, which was corresponded to 1/5 of that in the platelet enzyme. Microvessel lipoxygenase was inhibited by nordihydroguaiaretic acid but not by indomethacin.     

Concomitant suppression of plasma ACTH- and beta-endorphin-like immunoreactivity by cyproheptadine, naloxone, and somatostatin in the ectopic ACTH syndrome

Release of plasma ACTH- and beta-endorphin (beta-EP)-like immunoreactivity (LI) was studied in vivo in a patient with an ectopic ACTH-producing malignant thymoma. Administration of lysin vasopressin stimulated concomitant release of plasma ACTH- and beta-EP-LI. Administration of cyproheptadine, naloxone, and somatostatin significantly suppressed plasma levels of ACTH- and beta-EP-LI, while saline infusion did not. Gel exclusion chromatography of the plasma extracts revealed that ACTH-LI consisted of two components, large and small molecular weight form, while beta-EP-LI consisted of three components, large molecular weight, beta-lipotropin-, and beta-EP-sized form; each of these components was incompletely suppressed by somatostatin infusion. It is suggested that certain tumors may have acquired aberrant multiple receptors during malignant transformation which may lead to the paradoxical hormone response as demonstrated in this case.     

Covalent linkage of 125I-insulin to a cytosolic insulin-degrading enzyme

Cytosol extracts high in insulin-degrading activity were cross-linked to 125I-insulin with the bifunctional cross-linker disuccinimidyl suberate. With cytosols from either rat muscle, liver, kidney or brain or human erythrocytes, only a single protein (Mr = 110,000) was specifically labeled. Three different lines of evidence indicated that this labeled protein is insulin-degrading enzyme, a cysteine protease which accounts for most of the insulin-degrading activity in cell extracts. Firstly, the cross-linking of 125I-insulin to this protein is inhibited by unlabeled insulin over the same concentration range of insulin which inhibits degradation. Separated insulin A and B chain were less potent at inhibiting cross-linking, whereas bovine serum albumin and cytochrome c were without effect. Secondly, antibodies to purified insulin-degrading enzyme precipitated the labeled protein in parallel with their ability to precipitate the insulin-degrading activity of the extracts. Thirdly, when the insulin-degrading activity was purified 40,000-fold from erythrocytes, this Mr 110,000 protein co-purified. These results indicate that cross-linking 125I-insulin may be a convenient method for labeling the insulin-degrading enzyme.     

Structural changes of alpha2- and ovomacroglobulins

The plasma α₂-macroglobulin and its egg white homologue ovomacroglobulin were purified from several different species and their structure before and after the reaction with proteinases studied by electron microscopy. The negatively stained specimens showed either a ringlike structure or a flowerlike one before the reaction with proteinses, but their structures changed into open rectangular ones after the reaction. The translational frictional ratio f/f ₀ of human α2-macroglobulin and crocodilian ovomacroglobulin given in the literature is between 1.5 and 1.6 before and after the reaction with proteinases. The value reflects asymmetry due not to a high axial ratio, but rather to an openness of the structure resulting in a partially free draining character of the molecules. The computational method developed by Bloomfield and his co-workers based on the formalism of Kirkwood is used to calculate the frictional ratio of several models constructed from small spheres. The overall shape of the models is derived from electron micrographs. Although the degree of hydration is an unknown parameter in the calculation, reasonable agreement is obtained between the experimental values of f/f ₀ and the calculated ones. Combination of electron microscopic and hydrodynamic methods would be fruitful in the structural study of giant proteins such as α₂-macroglobulin.     

Cytogenetics of ticks (Acari: Ixodoidea)

Chromosomes and sex determination of 9 species of Haemaphysalis assigned to 4 subgenera are described. H. (tAlloceraea) kitaokai possesses an XX∶XO sex chromosome system with 18 autosomes plus XX in females; 18 plus X in males. H. (Kaiseriana) hystricis has 18 +XX and 18 + XY in females and males, respectively, in most specimens, but a supernumerary chromosome is present in some individuals. A supernumerary chromosome was also observed in 1 male H. (Aborphysalis) formosensis. These two species are the second and third species of ticks reported to have supernumerary chromosomes. H. formosensis, H. (Kaiseriana) bispinosa, H. (Haemaphysalis) campanulata, H. (H.) flava, H. (H.) megaspinosa, H. (H.) japonica, and H. (H.) pentalagi possess 20 autosomes plus 2 sex chromosomes in females and 20+1 sex chromosomes in males. Phylogenetic relationships within the genus Haemaphysalis are briefly discussed.     

Zymographic demonstration of lactate and malate dehydrogenases isoenzymes in the rodent salivary glands

Summary Analysis of lactate and malate dehydrogenase zymograms of rodent salivary glands showed species and organ specific patterns.Lactate dehydrogenase isoenzyme patterns occupied the middle positions in relation to those of skeletal and heart muscle. Activities of the major salivary glands were in the order submaxillary gland>parotid>sublingual gland. Zymogram of the mouse and rat showed LDH₄ and LDH₅ high activity patterns, while that of the rabbit was the fast moving active one. Hamster salivary gland exhibited a neutral type of the former and the latter.Malate dehydrogenase isoenzyme exhibited very similar patterns for the mouse, rat and hamster. Malate dehydrogenase zymogram of rabbit showed 3 active bands, which was different from the other rodents.     

The Large Isoform of Myelin-Associated Glycoprotein Is Scarcely Expressed in the Quaking Mouse Brain

Two polypeptide isoforms of myelin-associated glycoprotein (MAG) with molecular masses of 72 and 67 kDa are produced by alternative splicing of the exon 12 portion. Our previous work has demonstrated that in the quaking mouse brain this alternative splicing is lacking and that the mRNA coding the large MAG isoform (L-MAG) is scarcely expressed, whereas that of small MAG isoform (S-MAG) is overexpressed. In the present study, we prepared antisera specific to the S-MAG and L-MAG amino acid residues, respectively. Immunoblots showed that the L-MAG band was scarcely detectable in the quaking mouse brain, whereas the S-MAG band had an apparently higher molecular mass than in the normal control. Our immunohistochemical study also showed that L-MAG was scarcely stained in the quaking mouse brain. These results seemed to reflect a reduction in content of L-MAG mRNA and abnormal glycosylation in the quaking mouse brain.