Ribosomal protein S6 gene haploinsufficiency is associated with activation of a p53-dependent checkpoint during gastrulation

Nascent ribosome biogenesis is required during cell growth. To gain insight into the importance of this process during mouse oogenesis and embryonic development, we deleted one allele of the ribosomal protein S6 gene in growing oocytes and generated S6-heterozygous embryos. Oogenesis and embryonic development until embryonic day 5.5 (E5.5) were normal. However, inhibition of entry into M phase of the cell cycle and apoptosis became evident post-E5.5 and led to perigastrulation lethality. Genetic inactivation of p53 bypassed this checkpoint and prolonged development until E12.5, when the embryos died, showing decreased expression of D-type cyclins, diminished fetal liver erythropoiesis, and placental defects. Thus, a p53-dependent checkpoint is activated during gastrulation in response to ribosome insufficiency to prevent improper execution of the developmental program.     

Synthesis, cytostatic and anti-HIV evaluations of the new unsaturated acyclic C-5 pyrimidine nucleoside analogues

A series of the novel C-5 alkynyl pyrimidine nucleoside analogues (1-14) in which the sugar moiety was replaced by the conformationally restricted Z- and E-2-butenyl spacer between the phthalimido and pyrimidine ring were synthesized by using Sonogashira cross-coupling reaction. Cytostatic activity evaluation of the novel compounds showed that E-isomers exhibited, in general, better cytostatic activities than the corresponding Z-isomers. E-isomer 14 exhibited the best cytostatic effect against all evaluated malignant cell lines, particularly against hepatocellular carcinoma (Hep G2, IC(50)=4.3microM). However, this compound was also cytotoxic to human normal fibroblasts (WI 38). Its Z-isomer 7 showed highly specific antiproliferative activity against Hep G2 (IC(50)=18microM) and no cytotoxicity to WI 38. Moreover, compounds 3, 4 and 14 expressed some marginal inhibitory activity against HIV-1 and HIV-2.     

The influence of vitamin C supplementation on the oxidative status of rat interscapular brown adipose tissue

The interscapular brown adipose tissue (IBAT) thermogenesis is accompanied with oxidative stress. In spite of the ability of rats to synthesize vitamin C, we tested the possibility that its additional intake may improve the tissue antioxidative protection. Thus, we studied the IBAT oxidative status in rats supplemented by two doses of ascorbic acid over a 4-week period of time.

Our results confirmed that the additional intake of ascorbate improves the tissue antioxidative defense. Probably acting through enhanced insulin release, vitamin C also exerted some metabolic effects, which emphasize its role in the regulation of IBAT functions under normal physiological conditions.     

EGF receptor signalling protects smooth-cuticle cells from apoptosis during Drosophila ventral epidermis development

Patterning of the Drosophila ventral epidermis is a tractable model for understanding the role of signalling pathways in development. Interplay between Wingless and EGFR signalling determines the segmentally repeated pattern of alternating denticle belts and smooth cuticle: spitz group genes, which encode factors that stimulate EGFR signalling, induce the denticle fate, while Wingless signalling antagonizes the effect of EGFR signalling, allowing cells to adopt the smooth-cuticle fate. Medial fusion of denticle belts is also a hallmark of spitz group genes, yet its underlying cause is unknown. We have studied this phenotype and discovered a new function for EGFR signalling in epidermal patterning. Smooth-cuticle cells, which are receiving Wingless signalling, are nevertheless dependent on EGFR signalling for survival. Reducing EGFR signalling results in apoptosis of smooth-cuticle cells between stages 12 and 14, bringing adjacent denticle regions together to result in denticle belt fusions by stage 15. Multiple factors stimulate EGFR signalling to promote smooth-cuticle cell survival: in addition to the spitz group genes, Rhomboid-3/roughoid, but not Rhomboid-2 or -4, and the neuregulin-like ligand Vein also function in survival signalling. Pointed mutants display the lowest frequency of fusions, suggesting that EGFR signalling may inhibit apoptosis primarily at the post-translational level. All ventral epidermal cells therefore require some level of EGFR signalling; high levels specify the denticle fate, while lower levels maintain smooth-cuticle cell survival. This strategy might guard against developmental errors, and may be conserved in mammalian epidermal patterning.     

C2-symmetric inhibitors of Plasmodium falciparum plasmepsin II: synthesis and theoretical predictions

A series of C(2)-symmetric compounds with a mannitol-based scaffold has been investigated, both theoretically and experimentally, as Plm II inhibitors. Four different stereoisomers with either benzyloxy or allyloxy P1/P1' side chains were studied. Computational ranking of the binding affinities of the eight compounds was carried out using the linear interaction energy (LIE) method relying on a complex previously determined by crystallography. Within both series of isomers the theoretical binding energies were in agreement with the enzymatic measurements, illustrating the power of the LIE method for the prediction of ligand affinities prior to synthesis. The structural models of the enzyme-inhibitor complexes obtained from the MD simulations provided a basis for interpretation of further structure-activity relationships. Hence, the affinity of a structurally similar ligand, but with a different P2/P2' substituent was examined using the same procedure. The predicted improvement in binding constant agreed well with experimental results.     

Prevalence of Escherichia coli O157 in Saskatchewan Cattle: Characterization of Isolates by Using Random Amplified Polymorphic DNA PCR, Antibiotic Resistance Profiles, and Pathogenicity Determinants

The prevalence of Escherichia coli O157 associated with feedlot cattle in Saskatchewan was determined in a 10-month longitudinal study (3 feedlots) and a point prevalence study (20 feedlots). The prevalence of E. coli O157 at the three different sites in the horizontal study varied from 2.5 to 45%. The point prevalence of E. coli O157 among Saskatchewan cattle from 20 different feedlots ranged from 0% to a high of 57%. A statistically significant (P = 0.003) positive correlation was determined to exist between the density of cattle and the E. coli O157 prevalence rate. A significant correlation (P = 0.006) was also found between the E. coli O157 percent prevalence and the number of cattle housed/capacity ratio. All 194 E. coli O157 isolates obtained were highly virulent, and random amplified polymorphic DNA PCR analysis revealed that the isolates grouped into 39 different E. coli O157 subtypes, most of which were indigenous to specific feedlots. Two of the most predominant subtypes were detected in 11 different feedlots and formed distinct clusters in two geographic regions in the province. Antimicrobial susceptibility testing of the E. coli O157 isolates revealed that 10 were multidrug resistant and that 73 and 5 were resistant to sulfisoxazole and tetracycline, respectively.     

Geographic variation in the ecological effects of extinction of Australia's Pleistocene megafauna

Recent studies suggest that extinction of Pleistocene megafauna had large impacts on the structure and functioning of ecosystems, including increased fire and shifts in vegetation state. We argue that the ecological effects of mega‐herbivore extinction are likely to have varied geographically, and might have been reduced in environments of low productivity. We tested this at Caledonia Fen, a cool, high‐elevation site in southeast Australia with a palynological record reaching back approximately 140 ka. The dung fungus Sporormiella indicated that large herbivores were present through most of the early part of the last glacial cycle, but declined abruptly between 50–40 ka and did not recover. This event corresponds with evidence for continent‐wide extinction of Australia's Pleistocene megafauna at that time. An earlier episode of low Sporormiella occurrence coincided with evidence of raised water levels in the fen. Changes in wetland conditions can alter the accumulation of Sporormiella, but there was no such change when Sporormiella counts fell in the period 50–40 ka. We found no evidence that the decline in Sporormiella triggered increased fire or a change in vegetation, which remained a low grass/shrub steppe. This contrasts with a warmer and more humid site, Lynch's Crater in northeast Australia, where decline of dung fungi was followed by increased fire and transition from mixed sclerophyll forest and rainforest to uniform sclerophyll forest. Our results suggest that the magnitude of ecological responses to Pleistocene megafaunal extinction varied geographically, under the control of regional climates.     

Importance of the RpoE Regulon in Maintaining the Lipid Bilayer during Antimicrobial Treatment with the Polycationic Agent,Chlorhexidine

The emergence of multidrug resistance in bacteria has reached alarming levels. To solve this growing problem, discovery of novel cellular targets or pathways important for antimicrobial resistance is urgently needed. In this study, we explored how the alternative sigma factor, RpoE, protects Escherichia coli O157 against the toxic effects of the polycationic antimicrobial agent, chlorhexidine (CHX). Susceptibility of this organism to CHX was found to directly correlate to the growth rate, with the faster replicating wild‐type being more susceptible to CHX than its more slowly replicating ΔrpoE O157 mutant. Once the wild‐type and rpoE mutant strains had undergone growth arrest (entered the stationary growth phase), their resistance to CHX became entirely dependent on the functionality of RpoE. The RpoE regulon plays a critical role in maintaining the integrity of the asymmetric lipid bilayer of E. coli, thereby preventing the intracellular accumulation of CHX. Finally, using a single‐cell, high‐resolution, synchrotron‐based approach, we discovered a subpopulation of the rpoE mutant strain with no detectable intracellular CHX, a predominant characteristic of the wild‐type CHX‐resistant population. This finding reveals a role of phenotypic heterogeneity in antimicrobial resistance.     

Effects of dexrazoxane and amifostine on evolution of Doxorubicin cardiomyopathy in vivo

Doxorubicin is one of the most active drugs in oncology, with cardiotoxicity as a serious side effect of its application. The aim of this study was to investigate dexrazoxane and amifostine impact on the evolution of myocardial changes induced by doxorubicin. BalbC female mice were treated with doxorubicin only (10 mg/kg, single intravenous push), or with dexrazoxane (200 mg/kg, intraperitoneal [ip]) or amifostine (200 mg/kg, ip) 60 mins or 30 mins prior to treatment with doxorubicin, respectively. Blood sampling for determination of conventional serum-marker activity was performed 48 hrs later. The grade of histopathology changes was evaluated by light microscopy 1.5 and 3 months after treatments using the Billingham scoring method. Control groups consisted of nontreated mice. After doxorubicin-only treatment, the grade of heart tissue damage was found to increase in the period between 1.5 and 3 months. A similar but less intense progression was also detected in amifostine-pretreated animals, with significant difference among median Billingham scores between the two time points. The pretreatment with dexrazoxane suspended expansion of tissue lesions in time. Changes in serum enzyme activity revealed two correlations: the greater reduction in alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) leakage is associated with a lower percentage of damaged tissue, and the creatine kinase to alpha-HBDH percent of difference ratio being greater than one is correlated with limited spreading of pathological lesions. Our results indicate that the development of doxorubicin-induced heart failure is based on a slow and persistent expansion of pathological process even long after the completion of the treatment. Dexrazoxane has proved to be successful and superior over amifostine against such an evolution of doxorubicin cardiomyopathy.