Multiple polyadenylation sites in a Drosophila tropomyosin gene are used to generate functional mRNAs.

The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.     

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.     

Characterization of caspase processing and activation in HL-60 cell cytosol under cell-free conditions. Nucleotide requirement and inhibitor profile.

The present studies compared caspase activation under cell-free conditions in vitro and in etoposide-treated HL-60 leukemia cells in situ. Immunoblotting revealed that incubation of HL-60 cytosol at 30 degrees C in the presence of cytochrome c and ATP (or dATP) resulted in activation of procaspases-3, -6, and -7 but not -2 and -8. Although similar selectivity was observed in intact cells, affinity labeling revealed that the active caspase species generated in vitro and in situ differed in charge and abundance. ATP and dATP levels in intact HL-60 cells were higher than required for caspase activation in vitro and did not change before caspase activation in situ. Replacement of ATP with the poorly hydrolyzable analogs 5'-adenylyl methylenediphosphate, 5'-adenylyl imidodiphosphate, or 5'-adenylyl-O-(3-thiotriphos-phate) slowed caspase activation in vitro, suggesting that ATP hydrolysis is required. Caspase activation in vitro was insensitive to phosphatase and kinase inhibitors (okadaic acid, staurosporine, and genistein) but was inhibited by Zn(2+), aurintricarboxylic acid, and various protease inhibitors, including 3,4-dichloroisocoumarin, N(alpha)-p-tosyl-L-phenylalanine chloromethyl ketone, N(alpha)-p-tosyl-L-lysine chloromethyl ketone, and N-(N(alpha)-benzyloxycarbonylphenylalanyl)alanine fluoromethyl ketone, each of which inhibited recombinant caspases-3, -6, -7, and -9. Experiments with anti-neoepitope antiserum confirmed that these agents inhibited caspase-9 activation. Collectively, these results suggest that caspase-9 activation requires nucleotide hydrolysis and is inhibited by agents previously thought to affect apoptosis by other means.     

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.      

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.     

Femoral artery neointimal hyperplasia is reduced after wire injury in Ref-1+/- mice

Redox factor-1 (Ref-1) is a multifunctional protein that regulates redox, DNA repair, and the response to cell stress. We previously demonstrated that Ref-1(+/-) mice exhibit a significantly reduced Ref-1 mRNA and protein levels within the vasculature, which are associated with increased oxidative stress. The goal of this study was to test the hypothesis that partial loss of Ref-1 altered the cellular response to vascular injury. Fourteen days after femoral artery wire injury, we found that vessel intima-to-media ratio was significantly reduced in Ref-1(+/-) mice compared with that in wild-type mice (P < 0.01). Bromodeoxyuridine labeling and transferase-mediated dUTP nick-end labeling staining at 14 days did not differ in the Ref-1(+/-) mice. In vitro studies found no significant changes in either serum-induced proliferation or baseline apoptosis in Ref-1(+/-) vascular smooth muscle cells. Exposure to Fas ligand; however, did result in increased susceptibility of Ref-1(+/-) vascular smooth muscle cells to apoptosis (P < 0.001). Ref-1(+/-) mice exhibited an increase in circulating baseline levels of IL-10, IL-1alpha, and VEGF compared with those in wild-type mice but a marked impairment in these pathways in response to injury. In sum, loss of a single allele of Ref-1 is sufficient to reduce intimal lesion formation and to alter circulating cytokine and growth factor expression.     

Visfatin levels do not change after the oral glucose tolerance test and after a dexamethasone-induced increase in insulin resistance in humans

INTRODUCTION, MATERIAL AND METHODS: Visfatin is a cytokine, mainly expressed in visceral fat, that exerts insulin-mimicking effects in rodents through activation of an insulin receptor, although the binding-site is distinct from that of insulin. However, the mechanisms that regulate visfatin synthesis are still not fully understood. In particular, it is not clear whether short-term glucose-induced hyperglycaemia and hyperinsulinaemia as well as a glucocorticoid-induced increase in insulin resistance are reflected in appreciable alterations in serum visfatin levels in humans. In order to investigate this we measured serum visfatin, glucose and insulin concentrations during a 75.0 gram oral glucose tolerance test (OGTT) [Study 1], as well as before and after oral administration of dexamethasone [Study 2]. Study 1 included 17 subjects (2 males), aged 35.7 +/- 15.6 (mean +/- SD) years of BMI 35.2 +/- 9.3 kg/m(2). Blood samples were taken before (0 minutes) and at 60 and 120 minutes after glucose administration. Study 2 included 20 subjects (4 males, 5 subjects with type 2 diabetes), aged 42.1 +/- 17.2 years of BMI 36.7 +/- 8.38 kg/m(2) who underwent screening for Cushing's disease/syndrome. Dexamethasone was administered at a dose of 0.5 mg every 6 hours for 48 hours. Fasting serum concentrations of visfatin, glucose and insulin were assessed before (D0) and after 48 hours of dexamethasone administration (D2). Insulin resistance was assessed according to the HOMA method in non-diabetic individuals (n = 15). RESULTS: In Study 1 two subjects were found to have impaired glucose tolerance and one subject was found to have diabetes mellitus. Glucose administration resulted in a highly significant increase in insulin (from 11.4 +/- 7.2 microU/mL at 0 min to 98.9 +/- 68.6 microU/mL at 60 min and 72.6 +/- 45.1 microU/mL at 120 minute of OGTT, p < 0.001 for 60 and 120 minutes in comparison to baseline). However, there was no change in serum visfatin concentrations (84.6 +/- 11.6 ng/mL at 0 minutes, 82.6 +/- 12.7 ng/mL at 60 minutes and 81.1 +/- 14.5 ng/mL at 120 minutes of OGTT, p = ns). All subjects in Study 2 achieved suppression of cortisol concentrations below 50 nmo/l. Dexamethasone administration resulted in an increase in fasting insulin (from 11.5 +/- 6.9 to 16.9 +/- 7.6 microU/mL; p = 0.011) and an increase in HOMA (from 2.73 +/- 1.74 to 4.02 +/- 2.27; p = 0.015), albeit without a significant change in serum visfatin concentrations (61.1 +/- 19.8 vs. 68.3 +/- 19.4 ng/mL, p = ns). In neither Study 1 nor Study 2 was there any significant correlation between serum visfatin and age, BMI or HOMA. CONCLUSIONS: There is a striking difference between the marked rise in insulin concentrations and the lack of change in visfatin concentrations during the oral glucose tolerance test. This implies that it is highly unlikely that visfatin is involved in the short-term regulation of glucose homeostasis in human subjects. Dexamethasone administration (4 mg/48 hours) induces an increase in insulin resistance, although without significant change in serum visfatin concentrations. Therefore in contrast to the in vitro data, short term glucocorticoid administration does not result in appreciable changes in serum levels of this adipocytokine. Furthermore, the results of our study do not support the notion that glucocorticoid-induced insulin resistance is likely to be related to changes in serum concentrations of visfatin.     

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.      

Health-related quality of life in the patients on maintenance hemodialysis: the analysis of demographic and clinical factors

Health-related quality of life (HRQoL) among hemodialysis (HD) patients recently became a nephrologist's focus of interest. HRQoL is an important predictor of outcome in HD patients and need to be regularly assessed. The aim of the present study was to compare the HRQoL of chronic HD patients with general population and to analyze influencing sociodemographic and clinical factors. We included 255 prevalent HD patients from four dialysis centers. HRQoL was measured with The Medical Outcomes Study Short Form 36 Health Survey Questionnaire (SF-36). This data were compared with control group (N = 132) from the general Croatian population. Comparisons of SF-36 scale scores of HD patients regarding demographic and clinical factors (age, gender, education level, dialysis vintage and diabetes) were also performed and analyzed with a multivariate regression analysis. HRQoL in prevalent HD patients was relatively low (mean Physical Component Summary, PCS = 33.7, mean Mental Component Summary, MCS = 43.0) and was lower compared to the control group from the general population in all HRQoL domains, PCS and MCS scores. Almost 53% of the HD patients had the critical score PCS < 43 + MCS < 51 as the predictor of death and hospitalization. Better HRQoL was revealed in the patients < 65 years old, males, patients with higher educational level and in the patients on maintenance HD less than one year. Age was the only statistically significant predictor of PCS and MCS. Developments of HD technology, treatment of comorbidities, continuous patients' education, social and psychological support and use of other renal replacement modalities, especially kidney transplantation, may improve the HRQoL in these patients.