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.      

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.     

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.      

SAR and biological evaluation of novel trans-3,4-dimethyl-4-arylpiperidine derivatives as opioid antagonists

The phenolic hydroxy group of opiate-derived ligands is of known importance for biological activity. We have developed a SAR study around LY255582 by comparing the effect of the hydroxy group in the 2- and 4-position of the phenyl ring. Also, we have proved that the 3-position of the phenyl ring is optimal for opioid activity. Furthermore, we have successfully replaced the hydroxy group in LY255582 by carbamate and carboxamide groups. The new analogs have high affinity for the opioid receptors comparable to the corresponding phenol. Carboxamide analog 12 has an improved metabolism profile and proved to be efficacious in in vivo studies.     

Candidate genes and sensory functions in health and irritable bowel syndrome

Adrenergic and serotonergic (ADR-SER) mechanisms alter gut (GI) function; these effects are mediated through G protein transduction. Candidate genetic variations in ADR-SER were significantly associated with somatic scores in irritable bowel syndrome (IBS) and gastric emptying but not small bowel or colonic transit. Our aim was to assess whether candidate ADR-SER genes are associated with motor and sensory GI functions in IBS and subgroups on the basis of bowel dysfunction. In 122 patients with IBS and 39 healthy controls, we assessed gastrointestinal somatic symptoms and affect by validated questionnaires. We measured: gastric volume (GV), maximum tolerated volume, rectal compliance, sensation thresholds and ratings, and genetic variations including alpha2A (C-1291G), alpha2C (Del 332-325), GNbeta3 (C825T), and 5-HTTLPR. Demographics and genotype distributions were similar in the patients with IBS subgrouped on bowel function. There were significant associations between 5-HTTLPR SS genotype and absence of IBS symptoms and between 5-HTTLPR LS/SS genotype and increased rectal compliance and increased pain ratings, particularly at 12 and 24 mmHg distensions. GNbeta3 was associated only with fasting GV; we did not detect associations between alpha2A genotype and the gastrointestinal sensory or motor functions tested. We concluded that 5-HTTLPR LS/SS genotype is associated with both increased pain sensation and increased rectal compliance though the latter effect is unlikely to contribute to increased pain sensation ratings with LS/SS genotype. The data suggest the hypotheses that the endophenotype of visceral hypersensitivity in IBS may be partly related to genetic factors, and the association of GNbeta3 with fasting GV may explain, in part, the reported association of GNbeta3 with dyspepsia.     

Genetic variation in endocannabinoid metabolism, gastrointestinal motility, and sensation

Cannabinoid agonist inhibits gastrointestinal motility. The endocannabinoid, anandamide, is inactivated by fatty acid amide hydrolase (FAAH). A single nucleotide polymorphism in the human FAAH gene (C385A) reduces FAAH expression. Our aim was to evaluate associations between FAAH genotype variation and symptom phenotype, gastric emptying and volume, colonic transit, and rectal sensation in patients with functional gastrointestinal disorders (FGID). 482 FGID patients [Rome II positive, 159 constipation disorders, 184 diarrhea disorders (D-IBS), 86 mixed bowel function (M-IBS), 20 chronic abdominal pain (CAP), 33 functional dyspepsia], and 252 healthy volunteers (HV) underwent questionnaires and studies of phenotype and genotype from 2000 to 2007: 250 gastric emptying, 210 fasting and postprandial gastric volume, 152 colonic transit, and 123 rectal sensation. All had FAAH genotype [CC vs. polymorphic (CA/AA)] determined by TaqMan. FAAH genotype distribution of FGID patients and HV did not deviate from Hardy-Weinberg equilibrium. There was a significant association of FAAH genotype with FGID phenotype (overall chi(2), P = 0.011) and with specific individual phenotypes (P = 0.048). Thus FAAH CA/AA increases the odds (relative to HV) for D-IBS (P = 0.008), M-IBS (P = 0.012), and, possibly, CAP (P = 0.055). There was a significant association of FAAH CA/AA genotype with accelerated colonic transit in D-IBS (P = 0.037). There was no association of FAAH genotype with rectal sensation thresholds or ratings. The association of genetic variation in metabolism of endocannabinoids with symptom phenotype in D-IBS and M-IBS and with faster colonic transit in D-IBS supports the hypothesis that cannabinoid mechanisms may play a role in the control of colonic motility in humans and deserve further study.     

Releases of Norepinephrine and Dopamine in Ventriculocisternal Perfusions in Hepatectomized and Laparotomized Rats

Abstract: The completely hepatectomized rat has frequently been used as a model to study changes in the economy of norepinephrine (NE) and dopamine (DA) in hepatic coma. Hypothermia characteristically develops in hepatectomized rats and also occurs in patients in hepatic coma and is associated with improved survival in both. The aims of the present study were to measure both release and uptake of NE and release of DA in brain in warm (37°C) and cool (30–32°C) rats at 3–5 h after laparotomy or hepatectomy. Ventriculocisternal perfusions of the brain were performed on rats under basal conditions and during releases evoked by 40 m M K⁺. Basal releases of NE and DA and evoked release of DA were greater in the warm hepatectomized rats than in all other groups. In some studies, 10⁻⁵ M amitriptyline was added to the perfusates to assess whether neuronal uptake was changed after hepatectomy. Uptake of released NE was equally robust in cool hepatectomized as in cool laparotomized rats but could not be measured in warm hepatectomized rats because of amitriptyline toxicity in these rats. Decreases in NE and increases in DA content were found in most areas of the brain after perfusion. Increased releases of NE and DA may contribute to the pathogenesis of hepatic encephalopathy.