Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

Interferon-γ exerts its negative regulatory effect primarily on the earliest stages of murine erythroid progenitor cell development

Interferon-γ (INFγ) has been shown to suppress erythropoiesis and perhaps to contribute to the anemia of chronic disease. In this study we demonstrated that the concentration of INFγ required to suppress murine burst forming unit-erythroid (BFU-E) growth was significantly less than that required to suppress colony forming unit-erythroid (CFU-E) growth. INFγ acted at the most primitive step in erythroid progenitor cell differentiation and proliferation, as inhibition was maximal when added at the time of BFU-E culture initiation. Inhibition was progressively less if INF-γ addition was delayed after culture initiation. The effects of INFγ on BFU-E did not require the presence of interleukin-1α (IL-1α), tumor necrosis factor-α (TNFα), or granulocyte macrophage colony stimulating factor (GM-CSF), as its effects were not neutralized by monoclonal antibodies against IL-1α, TNFα, or GM-CSF. This applied whether INFγ was added to culture with individual antibodies or with a combination of all three antibodies. INFγ was not required for IL-1α- or TNFα-induced suppression of BFU-E, as their effects were not neutralized by a monoclonal anti-INFγ antibody. In contrast, GM-CSF—induced suppression of BFU-E was negated by the simultaneous addition of anti-INFγ. We have previously shown that the addition of TNFα does not suppress BFU-E growth in cultures from marrow depleted of macrophages. Suppression did occur, however, if a small concentration of INFγ that does not inhibit and increasing concentrations of TNFα were added to culture, suggesting a synergistic effect between INFγ and TNFα. These observations suggest that INFγ is a potent direct inhibitor of erythroid colony growth in vitro. It exerts its negative regulatory effect primarily on the earliest stages of erythroid progenitor cell differentiation and proliferation, as much higher doses are required to suppress late erythroid cell development. INFγ is also involved in GM-CSF—induced inhibition of BFU-E colony growth. © 1995 Wiley-Liss, Inc.

1 This artilce is a US Government work and, as such, is in the public domain in the United States of America.

     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Amino acid odorants stimulate microvillar sensory neurons

The olfactory epithelium (OE) of zebrafish is populated with ciliated and microvillar olfactory sensory neurons (OSNs). Whether distinct classes of odorants specifically activate either of these unique populations of OSNs is unknown. Previously we demonstrated that zebrafish OSNs could be labeled in an activity-dependent fashion by amino acid but not bile acid odorants. To determine which sensory neuron type was stimulated by amino acid odorants, we labeled OSNs using the ion channel permeant probe agmatine (AGB) and analyzed its distribution with conventional light- and electron-microscope immunocytochemical techniques. Approximately 7% of the sensory epithelium was labeled by AGB exposure alone. Following stimulation with one of the eight amino acids tested, the proportion of labeled epithelium increased from 9% for histidine to 19% for alanine; amino acid stimulated increases in labeling of 2-12% over control labeling. Only histidine failed to stimulate a significant increase in the proportion of labeled OSNs compared to control preparations. Most amino acid sensitive OSNs were located superficially in the epithelium and immuno-electron microscopy demonstrated that the labeled OSNs were predominantly microvillar. Large numbers of nanogold particles (20-60 per 1.5 microm(2)) were associated with microvillar olfactory sensory neurons (MSNs), while few such particles (<15 per 1.5 microm(2)) were observed over ciliated olfactory sensory neurons (CSNs), supporting cells (SCs) and areas without tissue, such as the lumen above the OE. Collectively, these findings indicate that microvillar sensory neurons are capable of detecting amino acid odorants.     

High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups

We recently proposed that patterns of evolution of non-LTR retrotransposable elements can be used to study patterns of spontaneous mutation. Transposition of non-LTR retrotransposable elements commonly results in creation of 5' truncated, "dead-on-arrival" copies. These inactive copies are effectively pseudogenes and, according to the neutral theory, their molecular evolution ought to reflect rates and patterns of spontaneous mutation. Maximum parsimony can be used to separate the evolution of active lineages of a non-LTR element from the fate of the "dead-on-arrival" insertions and to directly assess the relative frequencies of different types of spontaneous mutations. We applied this approach using a non-LTR element, Helena, in the Drosophila virilis group and have demonstrated a surprisingly high incidence of large deletions and the virtual absence of insertions. Based on these results, we suggested that Drosophila in general may exhibit a high rate of spontaneous large deletions and have hypothesized that such a high rate of DNA loss may help to explain the puzzling dearth of bona fide pseudogenes in Drosophila. We also speculated that variation in the rate of spontaneous deletion may contribute to the divergence of genome size in different taxa by affecting the amount of superfluous "junk" DNA such as, for example, pseudogenes or long introns. In this paper, we extend our analysis to the D. melanogaster subgroup, which last shared a common ancestor with the D. virilis group approximately 40 MYA. In a different region of the same transposable element, Helena, we demonstrate that inactive copies accumulate deletions in species of the D. melanogaster subgroup at a rate very similar to that of the D. virilis group. These results strongly suggest that the high rate of DNA loss is a general feature of Drosophila and not a peculiar property of a particular stretch of DNA in a particular species group.      

Oxytocin receptor binding in the hypothalamus during gestation in rats

Central oxytocin receptors (OTR) may be involved in adaptations of the brain oxytocin (OT) system during gestation, which are critical for systemic release of OT during parturition and lactation. We used quantitative autoradiography to determine changes in OTR binding in numerous brain sites during the course of gestation in the rat. Furthermore, to evaluate the importance of ovarian steroids in mediating pregnancy-related changes in OTR binding, we measured binding in ovariectomized animals treated with progesterone and/or estrogen, and in pregnant animals treated with exogenous progesterone during late gestation. We found that OTR binding was significantly increased in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) by midgestation (day 15) compared with control. In addition, there was a further significant increase in OTR binding in these nuclei by late gestation (day 20). The bed nucleus of the stria terminalis (BNST) and the medial preoptic area (MPOA) also showed significant gestation-associated increases in OTR binding, which were similar during mid- and late pregnancy. Treatment with exogenous progesterone throughout pregnancy did not alter the increase in OTR binding characteristic of late gestation in any of these brain sites. Finally, estrogen treatment in ovariectomized animals resulted in increased OTR binding in the SON, BNST, and MPOA, but not the PVN. These data demonstrate that OTR binding in the hypothalamus is increased during mid- and late-gestation, compared with ovariectomized control animals, which may be mediated by increased estradiol.