Synthesis of two novel thioacridinic derivatives and comparison of their in vitro biological activities.

Two novel compounds, 3-amino-9-(diethylaminoethylthio) acridine and 9-diethylaminoethylthioacridine, were synthesized and characterized. They were shown to be cytotoxic against K562 and Raji cell lines. A concentration of 10(-5) M killed around 40% of the cells after 3 h time of incubation. Intercalation into DNA was more efficient when a protonated nitrogen was present in a side chain of the ring system. At the cytotoxic concentrations (10(-5) M, 10(-6) M), inhibition of nucleic acid synthesis in K562, Raji cell lines and human leukocytes has been shown. The results presented suggest that the cytotoxicity and the inhibition of nucleic acid synthesis of the two compounds studied are inversely related to their intercalating capability into the DNA helix.     

Selective intracellular release of copper and zinc ions from bis(thiosemicarbazonato) complexes reduces levels of Alzheimer disease amyloid-beta peptide

Copper and zinc play important roles in Alzheimer disease pathology with recent reports describing potential therapeutics based on modulation of metal bioavailability. We examined the ability of a range of metal bis(thiosemicarbazonato) complexes (MII(btsc), where M=CuII or ZnII) to increase intracellular metal levels in Chinese hamster ovary cells overexpressing amyloid precursor protein (APP-CHO) and the subsequent effect on extracellular levels of amyloid-beta peptide (Abeta). The CuII(btsc) complexes were engineered to be either stable to both a change in oxidation state and dissociation of metal or susceptible to intracellular reduction and dissociation of metal. Treatment of APP-CHO cells with stable complexes resulted in elevated levels of intracellular copper with no effect on the detected levels of Abeta. Treatment with complexes susceptible to intracellular reduction increased intracellular copper levels but also resulted in a dose-dependent reduction in the levels of monomeric Abeta. Treatment with less stable ZnII(btsc) complexes increased intracellular zinc levels with a subsequent dose-dependent depletion of monomeric Abeta levels. The increased levels of intracellular bioavailable copper and zinc initiated a signaling cascade involving activation of phosphoinositol 3-kinase and c-Jun N-terminal kinase. Inhibition of these enzymes prevented Abeta depletion induced by the MII(btsc) complexes. Inhibition of metalloproteases also partially restored Abeta levels, implicating metal-driven metalloprotease activation in the extracellular monomeric Abeta depletion. However, a role for alternative metal-induced Abeta metabolism has not been ruled out. These studies demonstrate that MII(btsc) complexes have potential for Alzheimer disease therapy.     

A study to assess changes in myocardial perfusion after treatment with spinal cord stimulation and percutaneous myocardial laser revascularisation; data from a randomised trial

Background

What constitutes a "clinical trial" is inconsistently defined in the medical literature. With an initiative by Cancer Care Ontario (CCO) to report institutional clinical trials activity across the province of Ontario, Canada, we sought to investigate the variability in the interpretation of the term by local oncology professionals.

Methods

A survey amongst the physicians and nurses at the Juravinski Cancer Centre at Hamilton Health Sciences, Ontario was conducted. The survey included 12 summaries of local clinical research studies, and respondents were asked which they believed represented a clinical trial. Subsequently, they were asked which of the same 12 studies they believed should be labeled as clinical trials when considering separate definitions provided by CCO and by the Ontario Cancer Research Network (OCRN).

Results

A total of 66 (54%) of 123 surveys were completed; 32/46 (70%) by physicians, 21/59 (36%) by primary care nurses, and 13/18 (72%) by clinical trial nurses. Without a standardized definition, all studies, 12/12, were considered to be clinical trials by at least 50% of respondents. When provided with the CCO definition only 6/12 studies were considered to be clinical trials by the majority of respondents, while with the OCRN definition it was 9/12 studies. Studies evaluating natural health products, non-traditional medical interventions, and non-randomized studies with standard interventions consistently ranked the lowest, regardless of the definition used.

Conclusion

Oncology professionals appear to have a broadly inclusive baseline definition of what constitutes a clinical trial. Establishing rigor and consistency in the definition of a clinical trial is important for any program, institutional or jurisdictional based comparisons of clinical trials activity, especially when used as a quality indicator of patient care.     

Antibiotic production, accumulation of intracellular carbon reserves, and sporulation in Micromonospora echinospora (ATCC 15837)

The physiology of the actinomycete Micromonospora echinospora was examined during growth. Biphasic accumulation of glycogen occurred, initially during the early exponential growth phase, and again following the onset of sporulation at 120 h. Lipid levels increased during growth eventually representing 25% of the cell mass. A significant proportion of the lipid was found to be in the form of triacylglycerols, which were found to accumulate markedly during the sporulation phase. The disaccharide trehalose was also found to accumulate during growth with levels rising to 5% of the dry weight during the mycelial production phase, then remaining constant during sporulation. Antibiotic was produced transiently by the cultures over the period preceding sporulation.     

The acidic pin of RuvA modulates Holliday junction binding and processing by the RuvABC resolvasome

Holliday junctions are four-way branched DNA structures formed during recombination, replication and repair. They are processed in Escherichia coli by the RuvA, RuvB and RuvC proteins. RuvA targets the junction and facilitates loading of RuvB helicase and RuvC endonuclease to form complexes that catalyse junction branch migration (RuvAB) and resolution (RuvABC). We investigated the role of RuvA in these reactions and in particular the part played by the acidic pin located on its DNA-binding surface. By making appropriate substitutions of two key amino acids (Glu55 and Asp56), we altered the charge on the pin and investigated how this affected junction binding and processing. We show that two negative charges on each subunit of the pin are crucial. They facilitate junction targeting by preventing binding to duplex DNA and also constrain branch migration by RuvAB in a manner critical for junction processing. These findings provide the first direct evidence that RuvA has a mechanistic role in branch migration. They also provide insight into the coupling of branch migration and resolution by the RuvABC resolvasome.     

Environmental heterogeneity amplifies behavioural response to a temporal cycle

Resource acquisition is integral to maximise fitness, however in many ecosystems this requires adaptation to resource abundance and distributions that seldom stay constant. For predators, prey availability can vary at fine spatial and temporal scales as a result of changes in the physical environment, and therefore selection should favour individuals that can adapt their foraging behaviour accordingly. The tidal cycle is a short, yet predictable, temporal cycle, which can influence prey availability at temporal scales relevant to movement decisions. Here, we ask whether black‐legged kittiwakes Rissa tridactyla can adjust their foraging habitat selection according to the tidal cycle using GPS tracking studies at three sites of differing environmental heterogeneity. We used a hidden Markov model to classify kittiwake behaviour, and analysed habitat selection during foraging. As expected for a central‐place forager, we found that kittiwakes preferred to forage nearer to the breeding colony. However, we also show that habitat selection changed over the 12.4‐h tidal cycle, most likely because of changes in resource availability. Furthermore, we observed that environmental heterogeneity was associated with amplified changes in kittiwake habitat selection over the tidal cycle, potentially because environmental heterogeneity drives greater resource variation. Both predictable cycles and environmental heterogeneity are ubiquitous. Our results therefore suggest that, together, predictable cycles and environmental heterogeneity may shape predator behaviour across ecosystems.     

DNA structure specificity of Rap endonuclease.

The Rap protein of phage lambda is an endonuclease that nicks branched DNA structures. It has been proposed that Rap can nick D-loops formed during phage recombination to generate splice products without the need for the formation of a 4-strand (Holliday) junction. The structure specificity of Rap was investigated using a variety of branched DNA molecules made by annealing partially complementary oligo-nucleotides. On Holliday junctions, Rap endonuclease shows a requirement for magnesium or manganese ions, with Mn(2+)supporting 5-fold more cleavage than Mg(2+). The location of endonuclease incisions was determined on 3'-tailed D-loop, bubble, flayed duplex, 5'-flap and Y junction DNA substrates. In all cases, Rap preferentially cleaves at the branch point of these molecules. With a flayed duplex, incisions are made in the duplex adjacent to the single-strand arms. Comparison of binding and cleavage specificities revealed that Rap is highly structure-specific and exhibits a clear preference for 4- and 3-stranded DNA over Y and flayed duplex DNA. Almost no binding or cleavage was detected with duplex, partial duplex and single-stranded DNA. Thus Rap endonuclease shows a bias for structures that resemble D-loop and Holliday junction recombination intermediates.     

Characterization of a thermosensitive Escherichia coli aspartyl-tRNA synthetase mutant.

The Escherichia coli tls-1 strain carrying a mutated aspS gene (coding for aspartyl-tRNA synthetase), which causes a temperature-sensitive growth phenotype, was cloned by PCR, sequenced, and shown to contain a single mutation resulting in substitution by serine of the highly conserved proline 555, which is located in motif 3. When an aspS fragment spanning the codon for proline 555 was transformed into the tls-1 strain, it was shown to restore the wild-type phenotype via homologous recombination with the chromosomal tls-1 allele. The mutated AspRS purified from an overproducing strain displayed marked temperature sensitivity, with half-life values of 22 and 68 min (at 42 degrees C), respectively, for tRNA aminoacylation and ATP/PPi exchange activities. Km values for aspartic acid, ATP, and tRNA(Asp) did not significantly differ from those of the native enzyme; thus, mutation Pro555Ser lowers the stability of the functional configuration of both the acylation and the amino acid activation sites but has no significant effect on substrate binding. This decrease in stability appears to be related to a conformational change, as shown by gel filtration analysis. Structural data strongly suggest that the Pro555Ser mutation lowers the stability of the Lys556 and Thr557 positions, since these two residues, as shown by the crystallographic structure of the enzyme, are involved in the active site and in contacts with the tRNA acceptor arm, respectively.     

Cortical Mechanisms of Central Fatigue and Sense of Effort

The purpose of this study was to investigate cortical mechanisms upstream to the corticospinal motor neuron that may be associated with central fatigue and sense of effort during and after a fatigue task. We used two different isometric finger abduction protocols to examine the effects of muscle activation and fatigue the right first dorsal interosseous (FDI) of 12 participants. One protocol was intended to assess the effects of muscle activation with minimal fatigue (control) and the other was intended to elicit central fatigue (fatigue). We hypothesized that high frequency repetitive transcranial magnetic stimulation (rTMS) of the supplementary motor area (SMA) would hasten recovery from central fatigue and offset a fatigue-induced increase in sense of effort by facilitating the primary motor cortex (M1). Constant force-sensation contractions were used to assess sense of effort associated with muscle contraction. Paired-pulse TMS was used to assess intracortical inhibition (ICI) and facilitation (ICF) in the active M1 and interhemispheric inhibitory (IHI) was assessed to determine if compensation occurs via the resting M1. These measures were made during and after the muscle contraction protocols. Corticospinal excitability progressively declined with fatigue in the active hemisphere. ICF increased at task failure and ICI was also reduced at task failure with no changes in IHI found. Although fatigue is associated with progressive reductions in corticospinal excitability, compensatory changes in inhibition and facilitation may act within, but not between hemispheres of the M1. rTMS of the SMA following fatigue enhanced recovery of maximal voluntary force and higher levels of ICF were associated with lower sense of effort following stimulation. rTMS of the SMA may have reduced the amount of upstream drive required to maintain motor output, thus contributing to a lower sense of effort and increased rate of recovery of maximal force.