Evaluation of P16 expression in canine appendicular osteosarcoma.

BACKGROUND: Osteosarcoma (OSA) is a common malignant bone tumor of large breed dogs that occurs at predictable anatomic sites. At the time of initial diagnosis, most affected dogs have occult pulmonary metastases. Even with aggressive surgical treatment combined with chemotherapy, the majority of dogs diagnosed with OSA live less than 1 year from the time of diagnosis. The ability to identify canine OSA cases most responsive to treatment is needed. In humans, OSA is also an aggressive tumor that is histologically and molecularly similar to canine OSA. The expression of the tumor suppressor gene product P16 by human OSA tissue has been linked to a favorable response to chemotherapy. RESULTS: We identified an antibody that binds canine P16 and developed a canine OSA tissue microarray in order to test the hypothesis that P16 expression by canine OSA tissue is predictive of clinical outcome following amputation and chemotherapy. Although statistical significance was not reached, a trend was identified between the lack of canine OSA P16 expression and a shorter disease free interval. CONCLUSIONS: The identification of a molecular marker for canine OSA is an important goal and the results reported here justify a larger study.

An extremely luminous X-ray outburst at the birth of a supernova.

Massive stars end their short lives in spectacular explosions--supernovae--that synthesize new elements and drive galaxy evolution. Historically, supernovae were discovered mainly through their 'delayed' optical light (some days after the burst of neutrinos that marks the actual event), preventing observations in the first moments following the explosion. As a result, the progenitors of some supernovae and the events leading up to their violent demise remain intensely debated. Here we report the serendipitous discovery of a supernova at the time of the explosion, marked by an extremely luminous X-ray outburst. We attribute the outburst to the 'break-out' of the supernova shock wave from the progenitor star, and show that the inferred rate of such events agrees with that of all core-collapse supernovae. We predict that future wide-field X-ray surveys will catch each year hundreds of supernovae in the act of exploding.

Pilot randomised controlled trial of a radiation therapist-led educational intervention for breast cancer patients prior to commencing radiotherapy.

PURPOSE: Although patients receive information prior to commencing radiotherapy, they often experience anxiety and distress. We conducted a pilot randomised controlled trial to determine whether a radiation therapist led psycho-educational intervention for breast cancer patients prior to radiotherapy is likely to be effective in reducing radiotherapy-related concerns, patient anxiety and depression. METHODS: The intervention comprised two face-to-face consultations with a radiation therapist (one prior to radiation planning and the other prior to treatment). Patients completed surveys at baseline, prior to treatment planning and on the first day of treatment. Outcome measures included the Hospital Anxiety and Depression Scale, Radiation Therapy Related Patient Concerns and Radiation Therapy Knowledge Scales. RESULTS: One hundred and twenty two patients completed baseline measures. Fifty-eight patients received usual care, and 64 received the intervention. After the first consultation, patient anxiety was significantly lower in the intervention group (p = 0.048), as were concerns about radiotherapy (p = 0.001). There were no differences between groups for depression. Patient knowledge for the intervention group was higher after the first consultation (p < 0.001). CONCLUSION: This intervention is likely to be effective in reducing patient anxiety and concerns and increasing knowledge. Future research is required to test this intervention with a larger population.

TP53 mutations in canine brain tumors.

The p53 tumor suppressor gene (TP53) is the most frequently altered gene in human cancer. Mutation of the gene has been shown to be an important mechanism of p53 pathway inactivation in a variety of human brain tumors, particularly those of astrocytic origin. Genomic DNA from a series of 37 glial and 51 nonglial canine brain tumors was sequenced to determine the frequency of TP53 gene mutations involving exons 3-9. Exonic mutations were found in 3 of 88 tumors (3.4%) and specifically in 1 of 18 astrocytic tumors (5.5%). This is markedly lower than that reported in comparable human tumors, suggesting that alternative mechanisms of p53 inactivation are likely to be present if p53 function contributes significantly to oncogenesis in canine brain tumors.

Preparation of Pickering emulsions and colloidosomes with relatively narrow size distributions by stirred cell membrane emulsification.

Stirred cell membrane emulsification has been used to prepare Pickering emulsions and covalently cross-linked colloidosomes using poly(glycerol monomethacrylate) stabilized polystyrene particles as the sole emulsifier. Pickering emulsions of 44-269 µm in size can be prepared with coefficients of variation as low as 25%, by varying the emulsification parameters. The cell membranes consisted of 5 µm pores with a pore-to-pore spacing of 200 µm. Significantly more uniform emulsions are produced when these open pores are restricted to a narrow ring around the membrane surface. Increasing the oil flux rate through this annular ring membrane increases both the size and polydispersity of the resulting emulsion droplets. There was no evidence for a "push off" force contributing to droplet detachment over the oil flux range investigated. Increasing the paddle stirrer speed from 500 to 1500 rpm reduces the average droplet diameter from 269 to 51 µm while simultaneously decreasing the coefficient of variation from 47% to 25%. Any further increase in surface shear led to droplet breakup within the dispersion cell and resulted in a significantly more polydisperse emulsion. The Pickering emulsions reported here have much narrower droplet size distributions than those prepared in control experiments by conventional homogenization (25% vs 74% coefficients of variation). Finally, low polydispersity colloidosomes can be conveniently prepared by the addition of an oil soluble polymeric cross-linker to the dispersed phase to react with the stabilizer chains.

Polyamine-functional sterically stabilized latexes for covalently cross-linkable colloidosomes.

Sterically stabilized polystyrene latexes were prepared by aqueous emulsion polymerization using a poly(ethylene imine) (PEI) stabilizer in the presence of 4-vinylbenzyl chloride (4-VBC; 1.0 wt % based on styrene). Partial quaternization of the amine groups on the PEI chains by 4-VBC occurs in situ, hence producing a chemically grafted steric stabilizer. Such 4-VBC-modified PEI chains were grafted more efficiently onto the polystyrene particles than unmodified PEI, as judged by aqueous electrophoresis, XPS, and nitrogen microanalysis. Moreover, partially quaternized PEI gave significantly smaller polystyrene particles than those synthesized in the absence of any PEI stabilizer or those synthesized using unmodified PEI. The partially quaternized PEI-stabilized polystyrene latex proved to be an effective emulsifier at pH 9, forming stable oil-in-water Pickering emulsions when homogenized (12,000 rpm, 2 min, 20 °C) with four model oils, namely, n-dodecane, methyl myristate, isononyl isononanoate, and sunflower oil. The primary and/or secondary amine groups on the PEI stabilizer chains were successfully cross-linked using three commercially available polymeric reagents, namely, tolylene 2,4-diisocyanate-terminated poly(propylene glycol) (PPG-TDI), poly(propylene glycol) diglycidyl ether (PPG-DGE), or poly(ethylene glycol) diglycidyl ether (PEG-DGE). Cross-linking with the former reagent led to robust colloidosomes that survived the removal of the internal oil phase on washing with excess alcohol, as judged by optical microscopy and SEM. PPG-TDI reacted very rapidly with the PEI stabilizer chains, with cross-linking being achieved during homogenization. Well-defined colloidosomes could be formed only by using sunflower oil and isononyl isononanoate with this cross-linker at 20 °C. However, cooling to 0 °C allowed colloidosomes to be formed using n-dodecane, presumably because of the slower rate of cross-linking at this reduced temperature. PPG-DGE proved to be a more generic cross-linker because it formed robust colloidosomes with all four model oils. However, cross-linking was much slower than that achieved using PPG-TDI, with intact colloidosomes being formed only after ∼12 h at 20 °C. The PEG-DGE cross-linker allowed cross-linking to be conducted at 20 °C from the aqueous phase (rather from within the oil droplets for the oil-soluble PPG-TDI or PPG-DGE cross-linkers). In this case, well-defined colloidosomes were obtained at 50 vol % with surprisingly little intercolloidosome aggregation, as judged by laser diffraction studies.

CHARMM: the biomolecular simulation program.

CHARMM (Chemistry at HARvard Molecular Mechanics) is a highly versatile and widely used molecular simulation program. It has been developed over the last three decades with a primary focus on molecules of biological interest, including proteins, peptides, lipids, nucleic acids, carbohydrates, and small molecule ligands, as they occur in solution, crystals, and membrane environments. For the study of such systems, the program provides a large suite of computational tools that include numerous conformational and path sampling methods, free energy estimators, molecular minimization, dynamics, and analysis techniques, and model-building capabilities. The CHARMM program is applicable to problems involving a much broader class of many-particle systems. Calculations with CHARMM can be performed using a number of different energy functions and models, from mixed quantum mechanical-molecular mechanical force fields, to all-atom classical potential energy functions with explicit solvent and various boundary conditions, to implicit solvent and membrane models. The program has been ported to numerous platforms in both serial and parallel architectures. This article provides an overview of the program as it exists today with an emphasis on developments since the publication of the original CHARMM article in 1983.

Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus.

Oligonucleotide-directed mutagenesis of a cDNA encoding the hemagglutinin of influenza virus has been used to introduce single base changes into the sequence that codes for the conserved apolar "fusion peptide" at the amino-terminus of the HA2 subunit. The mutant sequences replaced the wild-type gene in SV40-HA recombinant virus vectors, and the altered HA proteins were expressed in simian cells. Three mutants have been constructed that introduce single, nonconservative amino acid changes in the fusion peptide, and three fusion phenotypes were observed: substitution of glutamic acid for the glycine residue at the amino-terminus of HA2 abolished all fusion activity; substitution of glutamic acid for the glycine residue at position 4 in HA2 raised the threshold pH and decreased the efficiency of fusion; and, finally, extension of the hydrophobic stretch by replacement of the glutamic acid at position 11 with glycine yielded a mutant protein that induced fusion of erythrocytes with cells with the same efficiency and pH profile as the wild-type protein. However, the ability of this mutant to induce polykaryon formation was greatly impaired. Nevertheless, all the mutant proteins underwent a pH-dependent conformational change and bound to liposomes. These results are discussed in terms of the mechanism of HA-induced membrane fusion.

Search for interstellar superheavy hydrogen.

Models for fundamental physical interactions allow for the existence of stable or nearly stable elementary particles much heavier than the proton. Stellar spectra were searched for a positively charged superheavy particle, X(+), which, with a bound electron, should appear as apparently superheavy neutral hydrogen in the interstellar medium. An upper limit for the abundance of X relative to normal hydrogen in the line of sight toward the bright star gamma Cassiopeiae is 2 x 10(-8).

Evaluation of a rapid immunodiagnostic test kit for detection of African lyssaviruses from brain material.

A rapid immunodiagnostic test kit was evaluated against a selection of isolates of lyssavirus genotypes occurring in Africa. The test was carried out in parallel comparison with the fluorescent antibody test (FAT) and isolates representing previously established phylogenetic groups from each genotype were included. The specificity of the rapid immunodiagnostic test compared favourably with the FAT and was found to detect all representatives of genotypes 1, 2, 3 and 4 in brain samples of either field cases or suckling mouse brain inoculates.

Enterostatin inhibition of angiogenesis: possible role of pAMPK and vascular endothelial growth factor A (VEGF-A).

INTRODUCTION: We utilized a genomic analysis of the response of neuronal GT1-7 cells to enterostatin to identify pathways responsive to this peptide. This information, together with reported properties of the enterostatin receptor, suggested that enterostatin may have an effect on angiogenesis. METHOD: To investigate this hypothesis, we studied the effect of enterostatin as an antiangiogenic agent in two angiogenic tissue culture model systems. RESULTS: Enterostatin induced a 50% or greater inhibition in the angiogenic response of human fat cells and had a U-shaped bimodal dose-response effect in inhibiting angiogenesis in a human placental vein angiogenesis model. To further understand this response, we tested enterostatin's effect in a human hepatoma cell line (HepG2 cells) that was subjected to glucose deprivation, a condition known to induce angiogenesis in other tumor cell lines. Phosphorylated AMP kinase (pAMPK) levels and vascular endothelial growth factor A (VEGF-A) mRNA expression were elevated robustly after incubation of HepG2 cells in the absence of glucose for 4 h, but 15 min incubation with enterostatin dramatically inhibited this pAMPK activation and reduced VEGF-A gene expression after 1 h incubation with enterostatin. The AMPK activator 5-aminoimidazole-4-carboximide ribonucleoside (AICAR) induced VEGF-A expression. SUMMARY: These data suggest that enterostatin has an antiangiogenic effect and suggest that it regulates VEGF-A gene expression through inhibition of AMPK activity.

The forkhead-associated domain protein Cep170 interacts with Polo-like kinase 1 and serves as a marker for mature centrioles.

We report the characterization of Cep170, a forkhead-associated (FHA) domain protein of previously unknown function. Cep170 was identified in a yeast two-hybrid screen for interactors of Polo-like kinase 1 (Plk1). In human cells, Cep170 is constantly expressed throughout the cell cycle but phosphorylated during mitosis. It interacts with Plk1 in vivo and can be phosphorylated by Plk1 in vitro, suggesting that it is a physiological substrate of this kinase. Both overexpression and small interfering RNA (siRNA)-mediated depletion studies suggest a role for Cep170 in microtuble organization and cell morphology. Cep170 associates with centrosomes during interphase and with spindle microtubules during mitosis. As shown by immunoelectron microscopy, Cep170 associates with subdistal appendages, typical of the mature mother centriole. Thus, anti-Cep170 antibodies stain only one centriole during G1, S, and early G2, but two centrioles during late G2 phase of the cell cycle. We show that Cep170 labeling can be used to discriminate bona fide centriole overduplication from centriole amplification that results from aborted cell division.

Expression and targeting of transcription factor ATF5 in dog gliomas.

BACKGROUND: Activating transcription factor 5 (ATF5) is a transcription factor that is highly expressed in undifferentiated neural progenitor/stem cells as well as a variety of human cancers including gliomas. AIMS: In this study, we examined the expression and localization of ATF5 protein in canine gliomas, and targeting of ATF5 function in canine glioma cell lines. MATERIALS AND METHODS: Paraffin-embedded canine brain glioma tissue sections and western blots of tumours and glioma cells were immunoassayed with anti-ATF5 antibody. Viability of glioma cells was tested with a synthetic cell-penetrating ATF5 peptide (CP-d/n ATF5) ATF5 antagonist. RESULTS: ATF5 protein expression was in the nucleus and cytoplasm and was present in normal adult brain and tumour samples, with significantly higher expression in tumours as shown by western immunoblotting. CP-d/n ATF5 was found to decrease cell viability in canine glioma cell lines in vitro in a dose-dependent manner. CONCLUSION: Similarities in expression of ATF5 in rodent, dog and human tumours, and cross species efficacy of the CP-d/n ATF5 peptide support the development of this ATF5-targeting approach as a novel and translational therapy in dog gliomas.

Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild-type p53.

Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity.

Molecular signalling pathways in canine gliomas.

In this study, we determined the expression of key signalling pathway proteins TP53, MDM2, P21, AKT, PTEN, RB1, P16, MTOR and MAPK in canine gliomas using western blotting. Protein expression was defined in three canine astrocytic glioma cell lines treated with CCNU, temozolamide or CPT-11 and was further evaluated in 22 spontaneous gliomas including high and low grade astrocytomas, high grade oligodendrogliomas and mixed oligoastrocytomas. Response to chemotherapeutic agents and cell survival were similar to that reported in human glioma cell lines. Alterations in expression of key human gliomagenesis pathway proteins were common in canine glioma tumour samples and segregated between oligodendroglial and astrocytic tumour types for some pathways. Both similarities and differences in protein expression were defined for canine gliomas compared to those reported in human tumour counterparts. The findings may inform more defined assessment of specific signalling pathways for targeted therapy of canine gliomas.

Somatosensory evoked potentials in man: differentiation of spinal pathways responsible for conduction from the forelimb vs hindlimb.

This review suggests that a previously unrecognized spinal cord pathway may be of major importance in the conduction of the somatosensory evoked potential (SEP) from the lower limb in Man. The nerve fiber type activated by a "typical" peripheral nerve stimulus used in studying the SEP will activate the posterior tibial nerve or median nerve predominantly at group I threshold. Group I fibers subserve limb proprioception. Therefore the spinal cord pathways subserving limb position sense will be the same pathways activated by a peripheral nerve stimulus used to evoke an SEP. A relatively newly recognized pathway involved with limb position sense from the lower limb is located in the dorsal portion of the lateral funiculus of the spinal cord whereas pathways subserving limb position sense in forelimb involve predominantly the dorsal column pathways. It is suggested that the dorsal columns play no major role in limb position sense from the lower limb in Man and therefore, the dorsal columns play no major role in the conduction of activity from the lower limb involved with the generation of an SEP.

Regulation of hepatic tyrosine aminotransferase in genetically obese rats.

The activities of hepatic tyrosine aminotransferase, tryptophan oxygenase and serine dehydratase were increased in obese rats shortly after weaning. Immunotitration experiments showed that the increase in tyrosine aminotransferase activity resulted from an increase in enzyme protein in obese rats. No increase in hepatic tyrosine aminotransferase was observed in suckling pre-obese rats. The post-weaning increase in hepatic tyrosine aminotransferase of obese rats was only observed during the light phase of the diurnal cycle, but was prevented by pair-feeding and by starvation. Tryptophan increased hepatic tyrosine aminotransferase of lean rats to obese levels but had no effect in obese rats until tyrosine aminotransferase levels were reduced by starvation or adrenalectomy. Adrenalectomy abolished the increase in hepatic tyrosine aminotransferase activity in obese rats although serum corticosterone was normal in these animals. Hepatic and brain tyrosine concentrations were decreased in obese rats but normalized after adrenalectomy. The results suggest that the corticosteroid-dependent increase in food and tryptophan intake may be the primary cause of the increased hepatic amino acid catabolism of obese rats.

Effects of insulin on the lipid structure of liver plasma membrane measured with fluorescence and ESR spectroscopic methods.

Insulin increased the lipid order of rat and mouse liver plasma membrane domains sampled by the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in a concentration-dependent saturable manner. The ordering is half maximal at 5.1 X 10(-11) M and fully saturated at 1.7 X 10(-10) M insulin. Membranes prepared from obese hyperglycemic (ob/ob) mice demonstrated a right-shift in the dose-dependent ordering induced by insulin, such that ordering was half maximal at 1.2 X 10(-10) M and fully saturated at 2.0 X 10(-10) M. Insulin also increased the order of rat liver plasma membranes labeled with the cis- and trans-parinaric acid methyl esters. The ordering caused by insulin as detected with cis methyl parinarate was complete within approx. 15 min. after hormone addition at 37 degrees C, and the ordering was approximately double that observed with the trans isomer. Additional ESR experiments demonstrated that the addition of insulin increased the outer hyperfine splittings of spectra recorded from membranes labeled with the steroid-like spin labels, nitroxide cholestane and nitroxide androstane, but not the fatty acid spin probe, 5-nitroxide stearate. Studies utilizing model membrane systems strongly suggest that the 5-nitroxide stearate samples a cholesterol-poor domain of the membrane, while the steroid-like probes preferentially sample cholesterol-rich regions of the membrane. Finally, insulin-induced membrane ordering was dose-dependently inhibited by cytochalasin B in the range 1-50 microM. From these results, we conclude that (1) the ordering effect of insulin addition to isolated liver plasma membrane fractions occurs within the physiological range of hormone concentration, and the dose-response is right-shifted in membranes from 'insulin resistant' animals; (2) the relative responses of the fluorescent and spin probes suggest that the effects of insulin are confined to specific domains within the membrane matrix; and (3) the direct effects of insulin on the membranes may involve protein components having cytochalasin B binding sites.

Fuel sensing and the central nervous system (CNS): implications for the regulation of energy balance and the treatment for obesity.

This review describes the product of the 3-day International Association for the Study of Obesity (IASO) Stock Conference held in March 2004 and sponsored by Abbott Laboratories. The conference was focused on how the mechanisms by which individual cells sense their own fuel status might influence the energy balance of the entire organism. Whether you are a single-celled organism or a sophisticated mammal with a large cerebral cortex, it is critical that cellular activity be matched to the available fuel necessary for that activity. Rapid progress has been made in the last decade in our understanding of the critical metabolic events that cells monitor to accomplish this critical task. More recent developments have begun to apply this understanding to how critical populations of neurones may monitor similar events to control both food intake and energy expenditure. The picture that emerges is that numerous peripheral fuel sensors communicate to the central nervous system (CNS) via neural and humoral routes. Moreover, it has been known for decades that specific populations of neurones sense changes in ambient glucose levels and adjust their firing rate in response and changes in neuronal glucose metabolism can influence energy balance. The CNS, however, does not just sense glucose but rather appears to be sensitive to a wide range of metabolic perturbations associated with fuel availability. This information is used to adjust both caloric intake and the disposition of fuels in the periphery. Increased understanding of these CNS fuel-sensing mechanisms may lead to novel therapeutic targets for obesity.

Effects of leptin on insulin secretion from isolated rat pancreatic islets.

Leptin is a hormone secreted by adipocytes as a peripheral metabolic signal for the central regulation of energy homeostasis or the reproductive system. Recent studies demonstrated that leptin receptor mRNA is expressed in pancreatic islets of rodents and that leptin at relatively high doses inhibits glucose-induced insulin secretion from rat islets. However, the physiological mechanism of leptin on insulin secretion has not been identified. In this study, we report that leptin inhibits glucose-induced insulin secretion at lower concentrations ranging from 25 to 50 ng/ml using a static incubation method. A perifusion study revealed that leptin (50 ng/ml) affected the second phase of insulin secretion but not the first phase. Leptin did not affect insulin secretion stimulated by glibenclamide (1 and 5 micromol/l) or forskolin (1 micromol/l). Leptin (50 ng/ml) significantly inhibited insulin secretion induced by the phorbol ester phorbol 12-myristate 13-acetate (TPA) in the presence of Ca2+ but not in the absence of Ca2+. Because TPA is known to activate protein kinase C (PKC), these present results suggest that leptin, at a physiological concentration, suppresses the second phase of insulin secretion by reducing activity of the Ca2+-dependent PKC isoform.