Studies on the adrenal cortex of hypophysectomized rats: A model for abnormal cellular atrophy and death

Summary Biochemical and ultrastructural studies indicate that the atrophy of adrenal cortex in hypoyhysectomized rats involves the following changes: (1) One to two days after hypophysectomy, there is loss of template activity resulting from cumulative DNA-damage and heterochromatinization.In vivo ACTH-administration led to recuperation of these cells, indicating damage during hypophysectomized state to be reversible. (2) If the duration of hypophysectomy is prolonged, some of the cells become irreversibly damaged and can no longer recuperate afterin vivo ACTH administration. (3) The period of most rapid cell death is from the third to seventh day after hypophysectomy. The cause of cell death is probably due to membrane damage in the absence of protein synthesis, leading to lysis of the cells. Lysozomes and macrophages are apparently not involved.Supported by U.S.P.H.S. grants AM-5384 and AM-13724 and taken in part from dissertations submitted by Chan and by Mostafapour to Wayne State University in partial fulfillment towards the Ph.D. degree.An invited article.     

Isolation and characterization of multiple forms of malt deoxyribonuclease

A deoxyribonuclease (DNase), similar to bovine pancreatic DNase, has been isolated from germinating barley. Commerically available malt was used as source of the enzyme. The purification procedure involves (a) ammonium sulfate fractionation (45–65% saturation), (b) CM-cellulose chromatography at pH 4.7 and (c) DEAE-cellulose chromatography at pH 8. DEAE-cellulose separates the enzyme into 4 distinct forms, designed as DNases A, B, C, and D. DNase A and B may be rechromatographed on DEAE-cellulose employing a CaCl₂ instead of Tris-HCl gradient. Both forms appear homogeneous on regular and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In addition, both forms have a sp. act. of ca 700 units per A unit at 280 nm, similar to the potency of the pancreatic enzyme. DNase C and D, which are present in relatively small quantities in malt, were not characterized. The MWs of DNases A and B, as estimated by the SDS gel electrophoresis techniques, are near 32 000, slightly larger than that of the pancreatic enzyme. In the presence of either Mn²⁺ or Mg²⁺, the pH-activity profile of the barley enzyme is similar to that obtained with the pancreatic enzyme. Like the pancreatic enzyme, barley DNase is protected by Ca²⁺ from inactivation. The amino acid compositions of the A and B forms are about the same; a comparison of the malt and pancreatic enzymes shows many similarities but major differences in the amounts of glutamic acid, proline and glycine. The hydrolysis products of DNA by malt DNase are indistinguishable from those obtained with pancreatic DNase. Further hydrolysis of these products by snake venom phosphodiesterase shows malt DNase to be a 5′-phosphate producer. Deoxythymidine 3′,5′-di-p-nitrophenyl phosphate, one of the synthetic substrates of pancreatic DNase, is also hydrolysed by malt DNase.     

Photosynthetic Pod Wall of Pea (Pisum sativum L.): Distribution of Carbon Dioxide-fixing Enzymes in Relation to Pod Structure

The pod wall of pea (Pisum sativum L.) was shown to contain two distinct photosynthetic layers. The outer, comprising chlorenchyma of the mesocarp, captured CO₂ from the outside atmosphere; the inner, a chloroplast-containing epidermis lining the pod gas cavity, was involved in photoassimilation of the CO₂ released from respiring seeds.     

Immunochemical studies on blood groups: The internal structure and immunological properties of water-soluble human blood group A substance studied by Smith degradation,liberation, and fractionation of oligosaccharides and reaction with lectins

Carbohydrate structures in the interior of a blood group A active substance (MSS) were exposed by one and by two Smith degradations. Reactivities of the original glycoprotein and its Smith degraded products with 13 different lectins and with anti-I Ma were studied by quantitative precipitin assay. MSS and its first Smith degraded product completely precipitated Ricinus communis hemagglutinin with five times less of the first Smith degraded glycoprotein being required for 50% precipitation. The second Smith degraded material precipitated only 90% of the lectin. MSS did not precipitate peanut lectin, whereas its first and second Smith degraded products completely precipitated the lectin. The first Smith degraded glycoprotein also reacted well with Wistaria floribunda, Maclura pomifera, Bauhinia purpurea alba, and Geodia lectins indicating that its carbohydrate moiety could contain dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc, dGalβ1 → 3dGlcNAcβ1 → 3dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc determinants at nonreducing ends. The second Smith degraded material precipitated well with Ricinus communis hemagglutinin, Arachis hypogaea, Geodia cydonium, Maclura pomifera, and Helix pomatia lectins showing that dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc residues at terminal nonreducing ends could be involved. Monoclonal anti-I Ma (group 1) serum reacted strongly with the first Smith degraded product indicating large numbers of anti-I Ma determinants, dGalβ1 → 4dGlcNAcβ1 → d 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc at nonreducing ends. The comparable activities of the native and Smith degraded products with wheat germ lectin indicate capacity to react with DGlcNAc residues at nonreducing ends and/or at positions in the interior of the chain. The totality of lectin reactivities indicates heterogeneity of the carbohydrate side chains. Oligosaccharides with ³H at their reducing ends released from the protein core of the first and second Smith degraded products were obtained by treatment with 0.05 m NaOH and 1 M NaB³H₄ at 50 °C for 16 h (Carlson degradation). The liberated reduced oligosaccharides were fractionated by dialysis, followed by retardion, Bio-Gel P-2, P-4, and P-6 columns. They were further purified on charcoal-celite columns, and by preparative paper chromatography and high-pressure liquid chromatography. Their distribution by size was estimated by the yields on dialysis, Bio-Gel P-2, and Bio-Gel P-6 chromatography, and from the radioactivity of the reduced sugars. Of the oligosaccharide fractions from the first Smith degraded product, about 77% of the carbohydrate side chain residues contained from 1 to 6 sugars, 13% from 7 to perhaps 12 sugars, and 10% was nondialyzable (polysaccharides and glycopeptide fragments). Of the second Smith degraded product, approximately 82% of carbohydrate residues had from 1 to 6 sugars, 14% from 7 to perhaps 20 sugars and 4% was nondialyzable. The biological activity profile of the two Smith degraded products together with the size distributions of the oligosaccharides indicated that their carbohydrate side chains, comprised a heterogeneous population ranging in size from 1 to about 12 sugars. When most of these chains that are shorter than hexasaccharides are fully characterized it may be possible to reconstruct the overall structure of the carbohydrate moiety of the blood group substances and account for their biological activities.     

Characterization of leukotriene A4 and B4 bioysnthesis

We have studied LTA₄ and LTB₄ synthesis in a cell-free system from RBL-1 cells. All the enzymes leading to the formation of LTB₄ from arachidonic acid are localized in the soluble fraction (100, 000 x g supernatant) of these cells. The formation of LTA₄ and LTB₄ is complete by 10 min. When we varied the arachidonic acid concentration from 1 to 300 μM, the synthesis of LTB₄ leveled off at 30 μM and of LTA₄ at 100 μM while 5-HETE had not reached a plateau at 300 μM. This enzyme system has the capacity to generate relatively large amounts of 5-HETE and LTA₄ and only a relatively small amount of LTB₄. Therefore, the rate limiting step is not the 5-lipoxygenase, the first step in the pathway, but the conversion of LTA₄ to LTB₄. This is in contrast to cyclooxygenase pathway where the first step is rate limiting. A second addition of arachidonic acid at submaximal concentration for LTA₄ synthesis did not produce any additional LTA₄ or LTB₄. Further study of this phenomenon showed that the 5-lipoxygenase and LTA-synthase were inactivated with time by preincubation with arachidonic acid and that peroxy fatty acids seem to be the inactivating species.     

Mitotic arrest and enhanced nuclear protein phosphorylation in human leukemia K562 cells by okadaic acid, a potent protein phosphatase inhibitor and tumor promoter.

We investigated the effects of the non-phorbol tumor promoter okadaic acid on human leukemia K562 cells. It was found that okadaic acid potently and reversibly inhibited cell growth, with a nearly complete inhibition of thymidine uptake seen at about 10 nM. The cytotoxicity of okadaic acid was characterized by a marked mitotic arrest of the cells exhibiting scattered chromosomes and abnormal anaphase-like structures, a phenomenon distinct from the typical metaphase arrest caused by colchicine. Okadaic acid (10-1,000 nM) greatly stimulated phosphorylation of a number of nuclear proteins in K562 cells. Phosphorylation of many of the same proteins was also stimulated by 12-O-tetradecanoylphorbol-13-O-acetate, a protein kinase C activator. The present findings, consistent with recent reports that okadaic acid is a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A) shown to be essential for normal mitosis, provided evidence for the first time that okadaic acid inhibition of PP1/PP2A resulted in enhanced nuclear protein phosphorylation and subsequent mitotic arrest.