Fluoride-inhibited calcium ATPase of sarcoplasmic reticulum. Magnesium and fluoride stoichiometry.

The sarcoplasmic reticulum (SR) CaATPase is inactivated by fluoride in the presence of magnesium (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 5229-5235). The inactive complex is very stable and can be isolated free of other components by 48 h of dialysis at 4 degrees C (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 16990-16994). In this study, we used a fluoride-specific electrode to determine that the amount of tightly bound fluoride in the complex was 9.4 +/- 2 nmol mg-1 SR protein. The rate constant of inactivation was very similar to the rate constant of fluoride incorporation and varied directly as the square of the fluoride concentration. Luminal Ca2+ accelerated reactivation of the inhibited enzyme, and the rate constants of activity regain and fluoride release were very similar. Although required for inhibition, added magnesium did not accelerate reactivation. Analysis for magnesium using antipyrylazo III of the inhibited enzyme showed 4.1 +/- 0.4 nmol mg-1 SR protein. As there is much evidence in the literature supportive of an estimate of calcium pumps equal to approximately 4-5 nmol mg-1 SR protein, our results indicate that each inhibited enzyme contains two tightly bound fluorides and one tightly bound magnesium.     

Defensive strategies of soft corals (Coelenterata: Octocorallia) of the Great Barrier Reef

Summary The relationship between ichthyotoxicity and predation-related defensive functional morphology was examined in alcyonacean soft corals of the central and northern regions of the Great Barrier Reef (GBR), Australia. Approximately 170 specimens were assessed encompassing a number of genera within three families: 1) the Alcyoniidae (Lobophytum, Sarcophytum, Sinularia, Cladiella, Parerythropodium, and Alcyonium); 2) Neptheidae (Lemnalia, Paralemnalia, Capnella, Lithophyton, Nephthea, Dendronephthya, Scleronephthya, and Stereonephthya), and 3) Xeniidae (Anthelia, Efflatounaria, Cespitularia, Heteroxenia, and Xenia). Ichthyotoxicity data were derived from earlier studies which used Gambusia affinis Baird and Girard (Vertebrata, Pisces) as a test organism. These data were compared to morphological data collected from specimens in the field and laboratory. Three sets of statistical analyses were performed, each considering a progressively narrower group of taxa. The first included 68 specimens and considered 16 morphological characters in each, falling into the general categories of gross colony form, colony texture, presence of mucus, colony color, polyp retractility, and sclerite morphology and distribution. These were tested for independence against ichthyotoxicity data. The second set of analyses involved a more restricted morphological data set derived from 28 species of Sinularia in combination with 28 species within the Nephtheidae, comparing them to their respective toxicity ranks. The third analysis considered the previous two taxonomic groups separately in relation to their toxicity levels.The attempt to consider many morphological characters in a taxonomically diverse collection did not reveal any general association in the Alcyonacea between defensive morphology and toxicity, and those associations which did emerge were clearly erroneous. The second analysis, considering only Sinularia spp. and nephtheids, demonstrated a negative association between ichthyotoxicity and the morphological characters of a) polypary armament, b) microarmament of the individual polyp, and c) strong mineralization of the coenenchyme. The third analysis revealed that the negative association found between toxicity and the first two characters was derived entirely from the nephtheids while the association detected between toxicity and the third character was restricted to Sinularia. It is concluded that a relationship between toxicity and morphology can be demonstrated, but it is heavily dependent upon which specific morphological characters are being considered and at what level of taxonomic resolution the analysis is being performed. An approach utilizing many characters over many taxa is unlikely to yield significant, reliable, or meaningful results.Australian Institute of Marine Science Contribution Number 383     

Induction of proliferation of neuroretina cells by long terminal repeat activation of the carboxy-terminal part of c-mil.

Expression of the P100gag-mil protein of avian retrovirus MH2 in cultured chicken embryo neuroretina cells was previously shown to result in the proliferation of normally quiescent cell populations. We show here that long terminal repeat activation of the carboxy terminus of the c-mil gene is sufficient to induce neuroretina cell proliferation.     

Trivalent behavior during prophase I in male mice heterozygous for three Robertsonian translocations: an electron-microscopic study

A synaptonemal complex (SC) analysis was carried out in male mice heterozygous (CHT/+) for three Robertsonian translocations. All pachytene preparations studied showed the presence of three trivalents. At early pachytene, the nonhomologous centromeric regions of the acrocentric chromosomes were unpaired. Heterosynapsis subsequently took place with complete pairing of the trivalents. Association between one of the three trivalents and the sex vesicle was observed in 30.4% of the nuclei. Association between the unpaired regions of two trivalents was present in 14.4% of the cells, suggesting that the relationship between unpaired regions of structural rearrangements and the X-Y bivalent may simply reflect the tendency of unpaired regions to establish end-to-end associations or heterosynapses among them, which are usually resolved during the pachytene stage of prophase I. Since the sex bivalent always has unpaired regions, these associations often affect the sex chromosomes.     

Structure of d(CACGTG), a Z-DNA hexamer containing AT base pairs.

The left-handed Z-DNA conformation has been observed in crystals made from the self-complementary DNA hexamer d(CACGTG). This is the first time that a non disordered Z form is found in the crystal structure of an alternating sequence containing AT base pairs without methylated or brominated cytosines. The structure has been determined and refined to an agreement factor R = 22.9% using 746 reflections in the resolution in the resolution shell 7 to 2.5 A. The overall shape of the molecule is very similar to the Z-structure of the related hexamer d(CG)3 confirming the rigidity of the Z form. No solvent molecules were detected in the minor groove of the helix near the A bases. The disruption of the spine of hydration in the AT step appears to be a general fact in the Z form in contrast with the B form. The biological relevance of the structure in relation to the CA genome repeats is discussed.     

Inhibition of skeletal muscle sarcoplasmic reticulum CaATPase activity by calmidazolium

Calmidazolium, a lipophilic cation and putative calmodulin-specific antagonist, inhibited potently the calcium ATPase of sarcoplasmic reticulum (SR) vesicles isolated from skeletal muscle. Based on steady-state measurements of catalytic activity over a range of MgATP, calmidazolium, and SR protein concentrations, the calculated values of the inhibition constant (KI) and binding stoichiometry were 0.06 microM and 770 nmol/mg protein, respectively. SR CaATPase inhibition apparently is not a general property of lipophilic cations since the hydrophobic anion tetraphenylboron inhibited catalysis, whereas its cationic analog, tetraphenylarsonium, did not. Enzyme inhibition by calmidazolium was noncompetitive with respect to the substrates Ca2+ and MgATP. In the presence of other SR CaATPase inhibitors, calmidazolium was competitive with respect to quercetin and noncompetitive with respect to trifluoperazine and propranolol. While calmidazolium inhibited enzyme phosphorylation by MgATP, catalysis was more sensitive to the inhibitor. Binding of calmidazolium to SR membranes produced morphological changes seen by electron microscopy as membrane thickening and loss of resolution of surface detail. Our results show that calmidazolium is a high-affinity, noncompetitive inhibitor of skeletal SR CaATPase activity, and they suggest that this inhibition is based on binding to the membrane phospholipids rather than specific antagonism of enzyme activation by calmodulin.     

v-mil induces autocrine growth and enhanced tumorigenicity in v-myc-transformed avian macrophages

MH2, an avian retrovirus containing the v-myc and v-mil oncogenes, rapidly transforms chick hematopoietic cells in vitro. The transformed cells belong to the macrophage lineage and proliferate in the absence of exogenous growth factors. Here we analyze a series of MH2 deletion mutants and show that these two oncogenes together establish an autocrine growth system in which v-myc stimulates cell proliferation, while v-mil induces the production of chicken myelomonocytic growth factor (cMGF). We also demonstrate that these two oncogenes cooperate in vivo. MH2 efficiently induces monocytic leukemias and liver tumors, while deletion mutants lacking either a functional v-mil or v-myc do not.     

Cytosolic free Ca2+ concentration and intracellular calcium distribution of Ca2+-tolerant isolated heart cells

The cytosolic free Ca2+ concentration of calcium-tolerant rat myocytes has been measured by the null point titration technique using arsenazo III as a Ca2+ indicator and digitonin to permeabilize the plasma membrane. The mean value obtained for 8 separate preparations was 270 +/- 35 nM. The distribution of releasable calcium between the mitochondrial and sarcoplasmic reticular compartments was measured by the successive additions of uncoupler and A23187 to cells pretreated with ruthenium red. The relative distribution of calcium in each pool was independent of the cell calcium content up to the maximum value of releasable calcium investigated (4.5 nmol/mg of cell dry weight) and was distributed in the approximate ratio of 2:1 in favor of the sarcoplasmic reticulum. The cells contained 1 nmol of calcium/mg of cell dry weight in a form nonreleasable by A23187, which was independent of the total cell calcium content as measured by atomic absorption spectroscopy. It is calculated that the calcium content of mitochondria in heart under physiological conditions is about 5 nmol/mg of mitochondrial protein. At this level, the mitochondria are likely to provide effective buffering of the cytosolic free Ca2+ concentration of quiescent heart cells. The corresponding intramitochondrial free Ca2+ is in a range above values needed to regulate the activity of Ca2+-dependent enzymes of the citric acid cycle in heart. The physiological calcium content of the sarcoplasmic reticulum in heart cells is estimated to be about 2.5 nmol/mg of cell dry weight, which is at least 5-fold greater than the amount of calcium release calculated to cause maximum tension development of cardiac muscle.     

Intraspecific Variation in Chemical Attraction of Rice to Insect Predators

The olfactory response of predators of the brown planthopper,Nilaparvata lugensStål, to different genotypes of rice (14 cultivars and breeding lines ofOryza sativaL. and 1 wild species,Oryza nivaraSharma et Shastry) was measured in an airflow olfactometer. Odor from rice plants attracted more females of the mirid predatorCyrtorhinus lividipennisReuter than plain air (control) on only 6 of the 15 rice genotypes. Orientation ofC. lividipennistoward volatiles of certain rice genotypes was apparent even when the plants were free of the brown planthopper. However, the predator distinguished between prey-infested and uninfested plants and preferred plants with eggs over plants with nymphs. The predator did not distinguish different stages of plant growth (vegetative, booting, or flowering). Plants artificially injured to simulate brown planthopper oviposition wounds were not as attractive to the predator as plants on which the planthopper had oviposited. The preassay preconditioning on the cultivar TN1 did not produce a predator bias for this genotype. This suggests that rearing effects or chemically mediated associative learning reported for some natural enemies did not influenceC. lividipennis'host response. Results with another predator, the coccinellidMicraspis hirashimaiSasaji, produced less consistent behavior. Planthopper-infested plants attracted more females ofM. hirashimaithan unifested plants in only 1 of the 12 rice genotypes evaluated. Implications for augmenting predators by rice cultivar selection and modification are discussed.