Biochemical Changes in Terminal Root Galls Caused by an Ectoparasitic Nematode,Longidorus africanus: Amino Acids

The amino acids of terminal root galls caused by Longidorus africanus on bur marigold (Bidens tripartita L.) and grapevine (Vitis vinifera L.) were studied. The galled roots of bur marigold contained 73% more cell-wall protein and 184% more free amino acids. The main changes among the free amino acids of the galled tissue were a large increase (1900%) in proline and a decrease in aspartic acid (56%) compared with the respective check tissue. Hydroxyproline decreased in the wall protein fraction from 5.6% in the healthy tissue to 3.6% in the infected tissue.Percent of hydroxyproline in total amino acids of the wall protein fraction of grapevine roots decreased from 0.7% in the healthy tissue to 0.3% in the galled tissue, and total proteins of this fraction decreased from 9.5 mg to 4.5 rag, respectively. Total protein in the protoplasmic fraction also decreased from 3.0 mg in healthy to 1.0 mg in infected roots. No change was noticed in total proteins in the free amino acids fraction but free proline decreased 40% in the infected roots.The relationship of these differences to the specific reactions of the hosts to nematode feeding is discussed.     

AUTOPHAGIC VACUOLES PRODUCED IN VITRO : II. Studies on the Mechanism of Formation of Autophagic Vacuoles Produced by Chloroquine

Continuous phase-contrast observations have been made on macrophages following exposure to chloroquine. The initial abnormality is the appearance in the Golgi region of small vacuoles with an intermediate density between that of pinosomes and granules. Over the course of 1–2 hr these vacuoles grow larger and accumulate amorphous material or lipid. Pinosomes or granules frequently fuse with the toxic vacuoles. Chloroquine derivatives can be seen by fluorescence microscopy; the drug is rapidly taken up by macrophages and localized in small foci in the Golgi region. Chloroquine continues to produce vacuoles when pinocytosis is suppressed. Electron microscopic studies of chloroquine effects on macrophages preincubated with colloidal gold to label predominately pinosomes or granules suggest that toxic vacuoles can arise from unlabeled organelles. Later vacuoles regularly acquire gold label, apparently by fusion, from both granules and pinosomes. L cells also develop autophagic vacuoles after exposure to chloroquine. Smooth endoplasmic reticulum apparently is involved early in the autophagic process in these cells. Information now available suggests an initial action of chloroquine on Golgi or smooth endoplasmic reticulum vesicles, and on granules, with alterations in their membranes leading to fusion with one another and with pinosomes.     

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : II. THE RELATION BETWEEN THE SOLUBILITY OF CASEIN AND ITS CAPACITY TO COMBINE WITH BASE. THE SOLUBILITY OF CASEIN IN SYSTEMS CONTAINING THE PROTEIN AND SODIUM HYDROXIDE.

1. The solubility in water of purified, uncombined casein has previously been reported to be 0.11 gm. in 1 liter at 25°C. This solubility represents the sum of the concentrations of the casein molecule and of the soluble ions into which it dissociates. 2. The solubility of casein has now been studied in systems containing the protein and varying amounts of sodium hydroxide. It was found that casein forms a well defined soluble disodium compound, and that solubility was completely determined by (a) the solubility of the casein molecule, and (b) the concentration of the disodium casein compound. 3. In our experiments each mol of sodium hydroxide combined with approximately 2,100 gm. of casein. 4. The equivalent combining weight of casein for this base is just half the minimal molecular weight as calculated from the sulfur and phosphorus content, and one-sixth the minimal molecular weight calculated from the tryptophane content of casein. 5. From the study of systems containing the protein and very small amounts of sodium hydroxide it was possible to determine the solubility of the casein molecule, and also the degree to which it dissociated as a divalent acid and combined with base. 6. Solubility in such systems increased in direct proportion to the amount of sodium hydroxide they contained. 7. The concentration of the soluble casein compound varied inversely as the square of the hydrogen ion concentration, directly as the solubility of the casein molecule, Su, and as the constants Ka₁ and Ka₂ defining its acid dissociation. 8. The product of the solubility of the casein molecule and its acid dissociation constants yields the solubility product constant, Su·Ka₁·Ka₂ = 2.2 x 10^–12 gm. casein per liter at 25°C. 9. The solubility of the casein molecule has been estimated from this constant, and also from the relation between the solubility of the casein and the sodium hydroxide concentration, to be approximately 0.09 gm. per liter at 25°C. 10. The product of the acid dissociation constants, Ka₁ and Ka₂, must therefore be 24 x 10^–12N. 11. It is believed that these constants completely characterize the solubility of casein in systems containing the protein and small amounts of sodium hydroxide.     

Sulfated secreted forms of bovine and porcine parathyroid chromogranin A (secretory protein-I)

Chromogranin A (secretory protein-I) is an acidic sulfated glycoprotein found in secretory granules of most endocrine and neuroendocrine cells. In the parathyroid it is co-stored and secreted with parathormone in response to hypocalcemia. Differences in post-translational modifications have been reported between chromogranin A from the bovine adrenal and porcine parathyroid glands. The former has been reported to be sulfated mainly on oligosaccharide residues and apparently includes a proteoglycan form, whereas the latter was previously reported to be tyrosine sulfated with little of the proteoglycan form present. Here we have directly compared 35SO4-labeled parathyroid chromogranin A from the pig and the cow to determine if these reported differences were tissue or species specific. We find that the chromogranin A secreted by the bovine gland contains a proteoglycan form, whereas that from the porcine gland does not. Moreover, chromogranin A of both species is primarily sulfated on oligosaccharide residues with little if any tyrosine sulfate detected. Differences were detected in the structure of sulfated O-linked oligosaccharides in bovine and porcine parathyroid chromogranin A.     

The distribution of DNA excision-repair sites in human diploid fibroblasts following ultraviolet irradiation

Using the technique for separating DNA fragments containing excision-repair sites from total genomic DNA as described in the previous paper (Cohn, S. M., and Lieberman, M. W. (1984) J. Biol. Chem. 259, 12456-12462), we have developed a method for directly determining the distribution of excision-repair sites in the genome. DNA was prepared from confluent, diploid human fibroblasts which had been irradiated with ultraviolet light and incubated in the presence of 5-bromo-2'-deoxyuridine (BrdUrd), repaired fragments were isolated, and the dependence of the fraction of total DNA fragments containing excision-repair sites on DNA fragment length was determined by electrophoretic analysis. The observed dependence was compared to the relationship expected for a random distribution of repair sites. At 36 h following 3 J/m2 UV, the distribution of repair sites was indistinguishable from a random distribution; however, at doses of UV above 6 J/m2, the observed dependence indicated that the distribution of repair sites was nonrandom. A time course of the distribution of repair sites following 12 J/m2 UV was clearly nonrandom from 4 h after irradiation until at least 36 h following irradiation. By 72 h, however, the distribution had become random. In cells treated with hydroxyurea, a reduced number of excision-repair sites were present, but the distribution of repair sites was also nonrandom. Autoradiographic analysis of the amount of unscheduled DNA synthesis in individual nuclei suggested that the nonrandom distribution of repair sites did not result from variable extents of repair synthesis in different cell populations or from cell death.     

The use of antibodies to 5-bromo-2'-deoxyuridine for the isolation of DNA sequences containing excision-repair sites

We have developed an immunological method for isolation and identification of DNA sequences containing 5-bromo-2'-deoxyuridine (BrdUrd) incorporated during UV-induced excision-repair synthesis. DNA fragments containing BrdUrd incorporated during repair synthesis were incubated with goat anti-BrdUrd and rabbit anti-goat IgG, and the antibody-DNA complexes were separated from bulk DNA by nitrocellulose filter binding. With this method, 80% of DNA sequences containing BrdUrd-labeled excision-repair sites were recovered, contaminated with less than 1% of DNA fragments devoid of excision-repair sites. Recovery of DNA fragments containing repair sites was independent of size from 2 to 20 kilobases. We have used this method in conjunction with blot hybridization to demonstrate that repair synthesis occurs in human ribosomal gene sequences in cells treated with UV.     

Nucleotide sequence comparison of the Adh gene in three drosophilids

Summary The alcohol dehydrogenase (Adh) gene has been isolated fromDrosophila simulans andD. mauritiana by screening clone libraries of each with a previously cloned Adh gene fromD. melanogaster. The isolated clones were subcloned and partially sequenced to determine the relatedness of these species and to examine details of evolutionary change in the structure of the Adh gene. We report the sequence of the first 704 nucleotides of each gene as well as 127 bases in the 5 untranslated region. When these sequences are compared,D. melanogaster differs fromD. simulans andD. mauritiana by 2.8% and 3.1%, respectively.D. simulans andD. mauritiana differ by only 1.8%, implying that they are more closely related to each other than either is toD. melanogaster. This is consistent with phylogenetic relationships established by a variety of genetic, biochemical, and morphological means and illustrates that DNA sequencing of a single gene may be used to assess the evolutionary relationships of species.     

Highly efficient purification of the 33-, 24-, and 18-kDa proteins in spinach photosystem II by butanol/water phase partitioning and high-performance liquid chromatography

The 33-, 24-, and 18-kDa proteins involved in photosynthetic oxygen evolution were purified from spinach photosystem II particles by butanol/water phase partitioning and high-performance liquid chromatography with a silica-based cation-exchange column. With this procedure a significant improvement was made in the time required for the purification and also in the amount and purity of the proteins. The N-terminal sequence of amino acid was determined for the purified proteins. Partial degradation of the proteins, which sometimes occurred in the purification, was not detected in the new procedure.