Ultracytochemical localization and microprobe quantitation of calcium stores in the insect oocyte.

Detection of calcium in the follicles of Galleria mellonella (Lepidoptera) was performed using two cytochemical methods. Calcium precipitation was obtained either with ammonium oxalate (AO) or with N,N-naphtaloylhydroxylamine (NHA). In both cases the X-ray "on line" analysis monitored the presence of calcium in the oocytes, which was correlated with the accumulation of yolk spheres. Concentration of calcium in oocytes filled with yolk and treated with AO amounted to 9 mmoles per 1,000 g tissue wet weight. This value is similar to that calculated previously for follicles untreated with any reagent and prepared for the analysis by the freeze-drying technique (Przelecka et al. 1980). Examination of the ultrastructure of oocytes treated with NHA revealed calcium precipitate at the follicular epithelium/oocyte interface, in endocytotic canaliculi and vesicles formed by the oocyte plasma membrane, in ooplasm, and in yolk spheres. In oocytes treated with AO, the calcium-precipitate intermingled with the precipitate produced by the osmium alone. The presumed cause of this phenomenon is discussed.

Displacement of cell-surface associated calcium inhibits phagocytosis and Ca-ATPase activity in amoeba.

Displacement of calcium from the cell surface region was observed in cells treated with either chlorpromazine or reserpine with chlorotetracycline being used as a calcium-fluorescent probe. The drugs also significantly inhibit the intensity of phagocytosis and Ca-ATPase activity. The possible role of Ca associated with the cell surface region in regulation of both phagocytosis and Ca-ATPase was discussed.

Ultrastructural alterations of the egg chambers of Galleria mellonella L. (Lepidoptera) developing after reserpine administration.

In the egg chambers of the wax moth Galleria mellonella L., developing after application of reserpine, a multilayered epithelium was observed through all the developmental stages. This epithelium consisted of "light" cells loaded with intracellular organelles. No "dark" cells, characteristic of the control material, were observed. All cells of the egg chambers of reserpine-treated insects were characterized mostly by an abundance of microtubules, strong vacuolation of cytoplasm, and dilation of intercellular and perinuclear spaces. Moreover, changes, such as malformation of nuclei and nucleoli, appearance of large glycogen deposits, occurrence of widened cisternae and canaliculi of rough endoplasmic reticulum (RER) connected with lipid droplets, and appearance of nonhomogeneous protein yolk spheres, were noted in the oocyte. The observed disturbances in the ultrastructure of the developing ovary are discussed in the aspect of a direct action of reserpine through the insect neurohormonal system, and of a possible indirect one by an impairment of calcium homeostasis.

Model investigations on the structure of heterogeneous nuclear ribonucleoproteins.

Protein HD40 , an RNA-helix destabilizing protein (Mr 40 000) is the major component of 30S heterogeneous nuclear ribonucleoprotein particles (hnRNP) from Artemia salina. The physical properties and the amino acid composition of HD40 are analogous to those of a group of well conserved core hnRNP proteins of higher eukaryotes. HD40 binds to and disrupts the secondary structure of single-stranded polynucleotides and forms extended nucleoprotein filaments at a stoichiometry of one protein per 12-15 nucleotides. The addition of a saturating amount of HD40 (one protein per 8 nucleotides) converts the filaments into bead-like complexes that are similar in properties and appearance to native hnRNP. To gain an understanding of the structure of hnRNP, we have used analytical ultracentrifugation and electron microscopy to investigate the structure of beaded complexes reconstituted from HD40 and poly(A)n of defined sizes. A complex containing 160 nucleotides forms a disc that is 3 nm high by 18 nm in diameter. At n less than 160 the complexes form sectors of the disc: 40 nucleotides give rise to a quarter of a disc, 80 nucleotides, half a disc, etc. At n greater than 160, the additional nucleoprotein elements may either initiate the formation of a second disc adjacent to the first or stack on top of the first disc to form a 6 nm high helix with a diameter of 18 nm. A single "bead" sediments at approximately 30S and contains an average of approximately 300 nucleotides and 1.8 turns of the helix. Native hnRNP particles from A. salina sediment at about 30S, have a diameter of 18-20 nm, and contain RNA fragments 180 to 400 nucleotides long.

Structure of complexes between a major protein of heterogeneous nuclear ribonucleoprotein particles and polyribonucleotides.

We have investigated the structure of complexes formed between a series of poly(A)n (n = 30 to 480) and HD40 (helix-destabilizing protein, molecular weight of 40,000), the major protein component of 30 S heterogeneous nuclear ribonucleoprotein particles (hnRNP) from the brine shrimp Artemia salina. Protein HD40 is similar to corresponding hnRNP proteins from higher eukaryotes and the complexes it forms with single-stranded nucleic acids are strikingly similar to the native "beads-on-a-string" structure of hnRNP. Using analytical ultracentrifugation and electron microscopy we find: (1) complexes formed between HD40 and long ribohomopolymers also have a beads-on-a-string structure, showing that the ability to form this structure is an inherent property of HD40, and is not dependent on any structural features of natural RNA; (2) complexes between HD40 and poly(A)160 form disks that are about 3 nm high by 18 nm in diameter and contain 20 HD40 molecules; (3) complexes of HD40 with poly(A)n with fewer than 160 nucleotides form sectors of a disk: 40 nucleotides give rise to a quarter of a disk, 80 nucleotides, half a disk, etc. The molecular weights increase with the size of poly(A)n at the rate of 5300 per nucleotide, a stoichiometry of eight nucleotides per HD40; (4) as the size of the poly(A)n increases beyond 160 nucleotides, the additional nucleoprotein elements may either initiate the formation of a second disk adjacent to the first or stack on top of the first disk to form a 6 nm high helix with a diameter of 18 nm. Based on these results, we propose that the existence of lateral protein-protein interactions that produce the basic 3 nm X 18 nm disk, combined with the marginal stability of the helix result in (a) interruptions of the helix that give rise to the beads-on-a-string appearance of the complexes, and (b) inherent heterogeneity of individual "beads" which may contain one or more turns of the helix. From measurements of HD40 complexes with coliphage MS2 RNA, phi X174 viral DNA as well as with the homopolymers, a bead is estimated to contain an average of approximately 300 nucleotides; approximately 1 X 8 turns of the helix.