Quality control and biophysical characterisation data of VanSA.

This data article presents the results from quality control experiments including N-terminal sequencing, SEC-MALS and Mass Spectrometry for purified VanSA used in experiments described in (Hughes et al., 2017) [1]; in addition to ligand interaction measurements and thermal melting curves of VanSA in the presence of screened ligands from circular dichroism measurements as well as UV-vis absorbance spectra for the binding interaction of VanSA in the presence of screened ligands.

Characterisation of the selective binding of antibiotics vancomycin and teicoplanin by the VanS receptor regulating type A vancomycin resistance in the enterococci.

A-type resistance towards "last-line" glycopeptide antibiotic vancomycin in the leading hospital acquired infectious agent, the enterococci, is the most common in the UK. Resistance is regulated by the VanRASA two-component system, comprising the histidine sensor kinase VanSA and the partner response regulator VanRA. The nature of the activating ligand for VanSA has not been identified, therefore this work sought to identify and characterise ligand(s) for VanSA. In vitro approaches were used to screen the structural and activity effects of a range of potential ligands with purified VanSA protein. Of the screened ligands (glycopeptide antibiotics vancomycin and teicoplanin, and peptidoglycan components N-acetylmuramic acid, D-Ala-D-Ala and Ala-D-y-Glu-Lys-D-Ala-D-Ala) only glycopeptide antibiotics vancomycin and teicoplanin were found to bind VanSA with different affinities (vancomycin 70µM; teicoplanin 30 and 170µM), and were proposed to bind via exposed aromatic residues tryptophan and tyrosine. Furthermore, binding of the antibiotics induced quicker, longer-lived phosphorylation states for VanSA, proposing them as activators of type A vancomycin resistance in the enterococci.

Anticipating the next meal using meal behavioral profiles: a hybrid model-based stochastic predictive control algorithm for T1DM.

Automatic control of Type 1 Diabetes Mellitus (T1DM) with subcutaneous (SC) measurement of glucose concentration and subcutaneous (SC) insulin infusion is of great interest within the diabetes technology research community. The main challenge with the so-called "SC-SC" route to control is sensing and actuation delay, which tends to either destabilize the system or inhibit the aggressiveness of the controller in responding to meals and exercise. Model predictive control (MPC) is one strategy for mitigating delay, where optimal insulin infusions can be given in anticipation of future meal disturbances. Unfortunately, exact prior knowledge of meals can only be assured in a clinical environment and uncertainty about when and if meals will arrive could lead to catastrophic outcomes. As a follow-on to our recent paper in the IFAC symposium on Biological and Medical Systems (MCBMS 2009), we develop a control law that can anticipate meals given a probabilistic description of the patient's eating behavior in the form of a random meal (behavioral) profile. Preclinical in silico trials using the oral glucose meal model of Dalla Man et al. show that the control strategy provides a convenient means of accounting for uncertain prior knowledge of meals without compromising patient safety, even in the event that anticipated meals are skipped.

Modulation of P-glycoprotein-mediated doxorubicin resistance in canine cell lines.

OBJECTIVE: To characterize the chemosensitivity of wild type and multidrug resistant canine cell lines and determine the relative potency of the P-glycoprotein (Pgp) modulators verapamil, tamoxifen and a cyclosporin-A analog (PSC833). METHODS: The dose required to reduce cell proliferation to 50% of control (ED50) for doxorubicin (DOX) and cisplatin was determined for canine cell lines 4TG11-50c, OS2.4wt, OS2.4DX and the human cell line MCF7/DX. The effect of Pgp chemomodulators on cytotoxicity was quantified by determining the dose modifying factor [DMF = (ED50 of Dox alone)/(ED50 of Dox + Modulator)]. Relative potency of modulators was defined as DMFMOD1/DMFMOD2. Pgp function was assessed by DiOC2 dye retention and by accumulation of DOX after chemomodulator addition. RESULTS: All cell lines were equally cisplatin sensitive but varied in doxorubicin resistance. PSC833 was 12X, 5X and 2X more potent than tamoxifen in 4TG11-50c, OS2.4WT and OS2.4DX, respectively. Dye retention was a better indicator of chemomodulator-enhanced cytotoxicity than was DOX accumulation. CONCLUSIONS: Pgp inhibition is cell line, modulator and concentration dependent but the cytotoxic potency of a modulator may be predicted by the extent of dye retention in canine drug resistant cell lines.

Characterization of canine MDR1 mRNA: its abundance in drug resistant cell lines and in vivo.

BACKGROUND: Overexpression of the MDR1 gene often contributes to antineoplastic drug resistance. The purpose of this study was to characterize the canine MDR1 mRNA homologue and evaluate its expression in both canine cell lines and lymphomas. MATERIALS AND METHODS: The abundance of the canine MDR1 transcript was assessed in three resistant cell lines and in pretreatment canine lymphoma using semi-quantitative RT/PCR. RESULTS: Canine transcript was 4.5 Kb with 93% sequence homology to human MDR1, and 90% homology to mouse and hamster equivalent genes. Increase in MDR1 transcript levels was observed in three progressively resistant canine cell lines. De novo MDR1 transcript expression was independent of response to therapy in dogs with lymphoma. CONCLUSIONS: We conclude that the canine MDR1 mRNA homologue is structurally similar to the human transcript. Expression of MDR1 mRNA correlates with in vitro drug sensitivity but does not correlate with in vivo doxorubicin sensitivity in canine lymphoma.

Modulation of doxorubicin concentration by cyclosporin A in brain and testicular barrier tissues expressing P-glycoprotein in rats.

P-glycoprotein (Pgp) is an inducible transmembrane protein that functions as an ATP-dependent efflux pump. Pgp is normally expressed in two types of cells: specialized epithelial cells with secretory/excretory functions (e.g., proximal renal tubules) and specialized endothelial cells (e.g., the capillary endothelial cells of the blood-brain barrier). In normal tissues, Pgp could exert a cytoprotective effect by facilitating excretion of drugs. It follows that inhibition of Pgp would alter the pharmacokinetics of drugs, like doxorubicin, in cells that express Pgp. The purpose of this study was to determine whether or not inhibition of Pgp by cyclosporin A (CsA) facilitated the transport of certain drugs across the blood tissue barriers of the brain and testes (barriers tissues expressing Pgp). 120 retired male breeder CD Fisher rats were randomly assigned to groups of 4 rats each. They were given either CsA, CsA vehicle, or saline followed by doxorubicin (Dox), cisplatin (CDDP), Evan's blue (EB), sodium fluorescein (NaF), or horseradish peroxidase (HRP). There was a CsA dose dependent increase in the tissue concentration of doxorubicin in brain and testes, but platinum (Pt) concentrations, derived from CDDP, were unaffected. Unlike CDDP, Dox, can be effluxed by Pgp. These increases in Dox concentrations were not due to altered vascular permeability as a result of CsA treatment as determined by lack of EB. NaF, or HRP in brain parenchyma. Modulation of Pgp function may prove to be useful for improving chemotherapy efficacy for patients with malignancies affecting tissues with blood-tissue barriers.

Ca(2+)-dependent nitric oxide release in endothelial but not R3230Ac rat mammary adenocarcinoma cells.

We have characterized the ability of several cell types associated with the microvasculature of solid tumors to release nitric oxide (NO.) in response to increases in cytosolic Ca2+ concentration ([Ca2+]c). EA.hy926 immortalized human umbilical vein endothelial cells (EC), rat fibroblasts (RFL), and tumorigenic cells isolated from R3230Ac rat mammary adenocarcinoma (MaC) were treated with thapsigargin (TG), an inhibitor of Ca(2+)-ATPase. NO. output was measured via a chemiluminescence detection system. Baseline NO. output was detectable only for EC. TG caused a significant increase in EC NO. output that could be blocked with NG-monomethyl-L-arginine and restored with L-arginine. TG did not stimulate NO. release from RFL or MaC cells, despite elevating [Ca2+]c in all cells. A Ca(2+)-dependent isoform of NO synthase (eNOS) was detected by immunoblot only in EC. These data indicate that EC, but not RFL or MaC, are capable of Ca(2+)-dependent NO. release and suggest that any Ca(2+)-dependent NO. release within this tumor is primarily of endothelial (and not tumorigenic cell) origin.

P-glycoprotein expression in canine lymphoma: a relevant, intermediate model of multidrug resistance.

BACKGROUND: Despite extensive investigation, the role of MDR of human cancer remains unclear. Canine lymphoma is a spontaneously arising correlate of human non-Hodgkin's lymphoma that may complement other in vivo models for investigation of issues related to MDR. METHODS: Immunoreactivity of primary antibodies to the human MDR1 gene product, p-glycoprotein 170 (Pgp), were determined in both a retrospective (n=76) and prospective (n=15) survey of canine lymphoma. Known prognostic factors and response to chemotherapy were correlated with categorical designations of Pgp expression. RESULTS: When combined, 61 of 91 samples (67%) were negative for Pgp, 16 of 91 (17.5%) had strong Pgp immunoreactivity in >50% of the malignant population and 14 of 91 (15.5%) had Pgp reactivity in 10-50% of cells. Pgp expression was greater after relapse compared with pretreatment samples [C494 83% vs. 25%; P=0.012 and C219 73% vs. 27%; P=0.04]. Pretreatment Pgp expression was an independent negative predictor of overall survival (median=225d vs. 367d; P=0.02). CONCLUSIONS: Pgp expression in spontaneous canine lymphoma is similar to that reported in human non-Hodgkin's lymphoma. Use of this model may expedite investigation of novel strategies for MDR prevention or modulation.

Overexpression of a p-glycoprotein in hepatocellular carcinomas from woodchuck hepatitis virus-infected woodchucks (Marmota monax).

The leading cause of human hepatocellular carcinomas (HCCs) is hepatitis B virus (HBV) infection. Woodchucks infected with a closely related hepadnavirus, woodchuck hepatitis virus (WHV), serve as a model for HBV because woodchucks chronically infected with WHV also develop hepatocellular carcinomas. Increased expression of p-glycoprotein (pgp) in human HCCs is a common obstacle in successful cancer chemotherapy. Pgps are encoded by a family of multidrug-resistance (MDR) genes. Livers from uninfected and WHV-infected woodchucks were examined to determine if pgp was expressed in HCCs and if there was a difference in expression between HCCs and nonneoplastic liver. A 170-kd protein was identified by Western blot in HCCs, whereas, constitutive pgp was not detected in normal liver taken from the same animals in 3 of 3 cases. Immunolocalization of the pgp with a panel of monoclonal antibodies revealed intensification of staining in 7 of 20 foci and 12 of 22 HCCs from six animals. Using primers for the human MDR1 gene, a single product was detected by reverse-transcribed polymerase chain reaction (RT-PCR) from HCCs. We have shown an increase in pgp in HCCs compared with normal liver from WHV-infected woodchucks. This is the first example of the induction of a pgp in a naturally hepadnavirus infected rodent system. It suggests the woodchuck can be a useful model for the study of the acquisition of resistance to chemotherapeutic agents in virally induced HCCs.

Cerebrospinal fluid collection in rats: modification of a previous technique.

Modification of a previously published technique for cerebrospinal fluid collection in rats is described. This technique uses general anesthesia, a supporting platform to flex the head-neck junction, a surgical approach to the dorsal atlanto-occipital region, and a micromanipulator to hold and control the approach of the collection needle. Critical steps for success of the procedure are correct positioning, avoiding premature incision of the subarachnoid space, and alignment of the collection needle with the midline and longitudinal axis of the head. The authors had a 95% success rate in obtaining > 0.1 ml of cerebrospinal fluid, using this technique.

Effects of mitomycin C and porfiromycin on exponentially growing and plateau phase cultures.

Laboratory studies and clinical trials are exploring the use of hypoxia-directed cytotoxic agents as adjuncts to radiotherapy. Because hypoxia and the microenvironmental inadequacies associated with hypoxia in solid tumours inhibit cell proliferation, an essential requirement for the successful use of hypoxia-directed drugs in cancer therapy is that these drugs be toxic to quiescent tumour cells, as well as tumour cells progressing rapidly through the cell cycle. The experiments reported here compared the cytotoxicities of mitomycin C and porfiromycin to exponentially growing and plateau phase cultures of EMT6 mouse mammary tumour cells. The proliferative status of the cultures did not influence the cytotoxicity of mitomycin C under either aerobic or hypoxic conditions, or the cytotoxicity of porfiromycin in air. Exponentially growing cultures were slightly more sensitive than plateau phase cultures to porfiromycin in hypoxia, but the difference between the sensitivities of proliferating and quiescent cells was much smaller than the difference between aerobic and hypoxic cells. No evidence for repair of potentially lethal damage was found after treatment with porfiromycin in air or in hypoxia; this is in agreement with previous findings for mitomycin C. Mitomycin C and porfiromycin therefore exhibit the toxicity to quiescent cells needed for effective use as hypoxia-directed drugs for the treatment of solid tumours.

Adducts of mitomycin C and DNA in EMT6 mouse mammary tumor cells: effects of hypoxia and dicumarol on adduct patterns.

6-CH3-3H-Mitomycin C (MC) was used to identify MC-DNA adducts formed in EMT6 mouse mammary tumor cells. DNA was isolated from cells treated with 3H-MC. The DNA was enzymatically digested, and the digest was analyzed for 3H-labeled adducts by high performance liquid chromatography. All four major adducts previously isolated and characterized in cell-free systems were detected: two different monoadducts and two bisadducts forming DNA-interstrand and DNA-intrastrand cross-links, respectively. No MC-DNA adducts other than the DNA interstrand cross-link had been shown previously to be formed in living cells. A MC-deoxyguanosine adduct of unknown structure was also detected in DNA from EMT6 cells; this adduct was also formed with purified EMT6 DNA. High performance liquid chromatography analysis was further applied to study the relationship between DNA adducts and cytotoxicity. The number of adducts increased with the concentration of MC in both aerobic and hypoxic cells. At a constant drug level, more adducts were observed in cells treated under hypoxic conditions than in cells treated aerobically; at 2 microM MC, 4.8 x 10(-7) and 3.1 x 10(-7) adducts/nucleotide were observed under hypoxic and aerobic conditions, respectively. The increased adduct frequency under hypoxia correlates with the known increased cytotoxicity of MC to EMT6 cells under hypoxic conditions. In addition, a higher ratio of cross-linked adducts to monoadducts was observed in hypoxic cells. The high performance liquid chromatography techniques were also used to examine the effects of dicumarol (DIC) on adduct patterns in cells treated simultaneously with 3H-MC. The MC-DNA adduct frequencies in DIC-treated cells were increased 1.5-fold under hypoxia and decreased 1.6-fold under aerobic conditions from those observed without DIC. This finding correlates with the known DIC-induced increase and decrease in the cytotoxicity of MC in hypoxic and aerobic EMT6 cells, respectively. The monoadduct resulting from monofunctionally activated MC was suppressed by DIC under both hypoxic and aerobic conditions. In addition, DIC induced the selective formation of an unknown DNA-associated radiolabeled substance in hypoxic cells; this is hypothesized to be a cytotoxic DNA lesion produced by a DIC-stimulated oxido-reductase. The methodology developed to measure MC adduct patterns may be useful as an indicator of distinct enzymatic activation processes for this drug.

Porfiromycin as an adjunct to radiotherapy in young and old mice.

Radiobiological data and measurements with O2 microelectrodes show that EMT6 tumors implanted into aged mice have a higher proportion of radioresistant, hypoxic cells than do tumors implanted into young adult animals; radiation is less effective in killing cells in tumors in old mice than in tumors in young adult mice. The studies reported here examine the effects of porfiromycin (POR), a bioreductive alkylating agent shown previously to be preferentially toxic to hypoxic EMT6 cells in vitro and in solid tumors in young adult mice. POR was effective in attacking the hypoxic cells of tumors in aged mice; regimens combining POR with x-rays overcame the radioresistance of tumors in the old animals. Comparisons of the distribution of 3H-labeled POR in young and old mice showed that tumors in aged mice had a slightly larger proportion of areas with necrotic features, which bound higher levels of tritiated POR than did healthy tumor regions without necrotic features. Studies of histology, lissamine green distributions, binding of tritiated POR, and radiation and POR cytotoxicity suggested that tumors in old mice contained a larger proportion of poorly perfused tumor cells, and that cells in these regions were resistant to radiation and sensitive to POR. Studies of the distribution of POR in normal tissues and of the toxicity of POR to bone marrow progenitor cells (CFU-GM) revealed no differences between young and old animals, showing that the differences observed in tumors reflected differences in the microenvironments within the tumors, rather than differences in the processing of drug in young and old animals.

Perinatal toxicology screening.

Accurate identification of substance abusing mothers and their infants is critical for appropriate medical management as well as the collection of accurate information on the effects of illicit drug use on perinatal morbidity, mortality, and long-term neurobehavioral outcome in the infants. This study examines the differences found using two methods for urine toxicology screening at the time of obstetrical admission to the hospital. The institution of universal screening identified significantly more women than were previously identified through the use of a risk-directed protocol (P less than .0001). Women identified using either protocol were significantly more likely than toxicology-negative women to have had poor prenatal care and to have smoked and used alcohol during pregnancy (P less than .001). In the population studied, the multiple criteria needed to accurately identify mothers with positive-toxicology screens would also include screening over one half of the toxicology-negative mothers.

Preclinical evaluation of Oxygent as an adjunct to radiotherapy.

These studies examine the potential value of a concentrated emulsion of perfluorooctylbromide (perflubron; Oxygent, Alliance Pharmaceutical Corp.) as an adjunct to radiotherapy. The effects of Oxygent on solid tumors were examined using EMT6 mammary tumors in BALB/c mice and BA1112 rhabdomyosarcomas in WAG/rij rats. Treatment with Oxygent plus O2, carbogen (95% O2/5% CO2), or hyperbaric oxygen (HBO) increased the effects of radiation on the tumors. Analyses of tumor cell survival curves and measurements of intratumor pO2 showed that this potentiation reflected an increase in the proportion of well-oxygenated tumor cells. Neither treatment of the animals with carbogen, O2, or HBO alone nor treatment of air-breathing rodents with Oxygent produced changes of similar magnitude. Treatment with a vehicle emulsion containing all the components of Oxygent except the perflubron did not alter tumor radiosensitivity, showing that tumor radiosensitization required the oxygen-transporting perfluorocarbon, and did not result from any biologic or physiologic effects of other components of the emulsion. These studies also examined the effects of Oxygent on the radiation responses of mouse skin and bone marrow. Oxygent selectively increased the radiation sensitivity of tumors relative to these normal tissues, thereby increasing the therapeutic ratio and producing therapeutic gain. Oxygent appears to warrant further testing as an adjunct to cancer therapy.

Effects of hyperbaric oxygen and a perfluorooctylbromide emulsion on the radiation responses of tumors and normal tissues in rodents.

Perfluorochemical emulsions are being examined in many laboratory and clinical studies as possible adjuncts to radiotherapy and chemotherapy. The studies reported here examine the clinical potential of hyperbaric oxygen (HBO) in combination with a highly concentrated perfluorochemical emulsion (Oxygent) containing 100% w/v perfluorooctylbromide (PFOB). HBO alone produced only a small improvement in the radiation response of BA1112 tumors in WAG/rij rats, while regimens combining HBO with Oxygent produced much greater radiation sensitization. A sham emulsion, formulated without the O2-carrying PFOB, did not alter the radiation response of the tumors in comparison with that seen with HBO alone. Neither HBO nor Oxygent plus HBO altered the radiosensitivity of bone marrow progenitor cells in BALB/c mice. HBO alone augmented skin reactions in BALB/c mice, but addition of Oxygent did not alter the skin reactions in comparison to those seen with HBO alone. Regimens combining Oxygent with HBO selectively increased the radiation sensitivity of tumors relative to normal tissues, thereby enhancing the therapeutic ratio. These results support the potential usefulness of perfluorochemical emulsions and HBO in clinical radiation therapy.

Modulation of tumor oxygenation and radiosensitivity by a perfluorooctylbromide emulsion.

The effect of a concentrated perfluorooctylbromide emulsion (Oxygent) on the radiosensitivity and oxygenation of solid tumors was examined using EMT6 mammary tumors in BALB/c mice and BA1112 rhabdomyosarcomas in WAG/rij rats. Treatment with Oxygent plus carbogen or oxygen breathing increased the radiosensitivity of both tumors. Analysis of tumor cell survival data and polarographic measurements of intratumoral pO2 indicated that this potentiation reflected an increase in the proportion of well-oxygenated tumor cells. Treatments with carbogen breathing alone, with Oxygent plus air-breathing, or with a vehicle emulsion containing all the components except the perfluorocarbon did not produce comparable improvements in tumor radiosensitivity. Concentrated perfluorooctylbromide emulsions appear to warrant further development and preclinical testing as adjuncts to cancer therapy.

Survey of field outbreaks of avian infectious laryngotracheitis in England and Wales.

Field outbreaks of infectious laryngotracheitis in commercial chicken flocks in England and Wales between 1985 and 1988 were investigated. Material from 49 outbreaks was submitted to Liverpool University, and virus was isolated from 17 of them. The results of a questionnaire on each outbreak are described. Generally, the disease was of moderate severity, and mainly affected laying flocks; it occurred in birds of a wide age range but most of the outbreaks were in birds between 10 and 20 weeks of age. The disease was not seen more frequently at any particular time of the year, and there was no evidence of a common source of infection. Three of the affected flocks had recently been moved and were beginning to lay; these stresses may have caused the re-excretion of latent virus.

Induction of heat-shock proteins by glutamine. The 'feeding effect'.

Subconfluent, log-phase Chinese hamster ovary cells induced the major heat-shock proteins (hsp) when cells were refed, 40 hours after seeding. This method of inducing heat-shock proteins was also obtained by refeeding with fresh serum-free media, but not with media with a long shelf life or with media prepared without glutamine. It was observed that addition of glutamine alone to cultures at 40 hours post-seeding induced heat-shock proteins. Addition of ammonium chloride, however, had no discernible effect on heat-shock protein synthesis. Northern blot analysis indicated that this phenomenon reflected an increase in the levels of message for the constitutive/inducible member of the hsp 70 family, but not the non-constitutive member. To determine the effect of this induction on heat sensitivity, unfed and 'heat-shock-induced' refed cultures were heated at 45 degrees C. No significant difference in cell survival was observed. Therefore glutamine is the necessary ingredient required for the induction of heat-shock proteins and this method of inducing heat-shock proteins does not alter heat sensitivity.

Isolation, identification, and assay of [3H]-porfiromycin adducts of EMT6 mouse mammary tumor cell DNA: effects of hypoxia and dicumarol on adduct patterns.

[3H]-(N-la-methyl) Porfiromycin (POR) was employed to detect and identify the radiolabeled mono- and bis-adducts formed in living EMT6 mouse mammary tumor cells under different conditions. To provide authentic standard adducts, calf-thymus DNA was treated with POR under reductive activation, then digested to nucleosides and POR-nucleoside adducts. The three major adducts formed were isolated by HPLC and authenticated. Two were mono-adducts, composed of deoxyguanosine linked at its N2-position to C-1 of POR and of 10-decarbamoyl POR. The third was a bis-adduct, in which POR was crosslinked to two deoxyguanosines at their N2-positions. DNA from [3H]-POR treated EMT6 cells was digested an analyzed by HPLC. DNA-associated label was located in thymidine and in two mono-adducts and one bis-adduct identical to those described above. Label in thymidine resulted from N-demethylation of POR and reincorporation of label into new thymidylate residues. Adducts were formed more abundantly in hypoxia than in air. In addition, the mono-adduct to crosslink ratios were different, approximately 1:1 and 2:1 for hypoxic and aerobic cells, respectively. The different patterns of alkylation in air and hypoxia may be related to the greater toxicity of POR in hypoxia. When cells were treated simultaneously with POR and dicumarol, adduct levels were lower, and a new, unknown adduct was observed primarily under hypoxia; these changes may be related to the altered toxicity of POR in the presence of dicumarol. The HPLC assay detected simultaneously the full array of stable mono- and bis-adducts in DNA with good sensitivity (greater than or equal to 2 x 10(6) adducts/nucleotide) and excellent reproducibility. This assay should be generally applicable to all cells and tissues when MC or POR with high specific radioactivity can be employed.