Disconnected by design: analytic approach in treatment networks having no common comparator.

In a network meta-analysis, comparators of interest are ideally connected either directly or via one or more common comparators. However, in some therapeutic areas, the evidence base can produce networks that are disconnected, in which there is neither direct evidence nor an indirect route for comparing certain treatments within the network. Disconnected networks may occur when there is no accepted standard of care, when there has been a major paradigm shift in treatment, when use of a standard of care or placebo is debated, when a product receives orphan drug designation, or when there is a large number of available treatments and many accepted standards of care. These networks pose a challenge to decision makers and clinicians who want to estimate the relative efficacy and safety of newly available agents against alternatives. A currently recommended approach is to insert a distribution for the unknown treatment effect(s) into a network meta-analysis model of treatment effect. In this paper, we describe this approach along with two alternative Bayesian models that can accommodate disconnected networks. Additionally, we present a theoretical framework to guide the choice between modeling approaches. This paper presents researchers with the tools and framework for selecting appropriate models for indirect comparison of treatment efficacies when challenged with a disconnected framework. Copyright © 2016 John Wiley & Sons, Ltd.

Attachment of bovine parvovirus to O-linked alpha 2,3 neuraminic acid on glycophorin A.

The bovine parvovirus (BPV) hemagglutinates human erythrocytes by binding to glycophorin A (GPA). The purpose of this study was to determine which carbohydrate on GPA binds BPV. Treatment of GPA with alpha2,3,-6,-8 neuraminidase eliminated binding of BPV to GPA. Beta-elimination of O-linked sialic acids on GPA eliminated binding, while removal of N-linked carbohydrates using the N-glycosidase PNGase F failed to eliminate binding. Treatment of GPA with a neuraminidase which specifically cleaved alpha2,3 glycosidic bonds eliminated BPV binding and, following this treatment, virus binding to GPA was restored by reconstitution of alpha2,3-linked neuraminic acids. These results indicated the O-linked alpha2,3 neuraminic acids of GPA bind BPV.

DNA topoisomerase II as the target for the anticancer drug TOP-53: mechanistic basis for drug action.

TOP-53 is a promising anticancer agent that displays high activity against non-small cell lung cancer in animal tumor models [Utsugi, T., et al. (1996) Cancer Res. 56, 2809-2814]. Compared to its parent compound, etoposide, TOP-53 is considerably more toxic to non-small cell lung cancer cells, is more active at generating chromosomal breaks, and displays improved cellular uptake and pharmacokinetics in animal lung tissues. Despite the preclinical success of TOP-53, several questions remain regarding its cytotoxic mechanism. Therefore, this study characterized the basis for drug action. Results indicate that topoisomerase II is the primary cytotoxic target for TOP-53. Furthermore, the drug kills cells by acting as a topoisomerase II poison. TOP-53 exhibits a DNA cleavage site specificity that is identical to that of etoposide. Like its parent compound, the drug increases the number of enzyme-mediated DNA breaks by interfering with the DNA religation activity of the enzyme. TOP-53 is considerably more efficient than etoposide at enhancing topoisomerase II-mediated DNA cleavage and exhibits high activity against human topoisomerase IIalpha and IIbeta in vitro and in cultured cells. Therefore, at least in part, the enhanced cytotoxic activity of TOP-53 can be attributed to an enhanced activity against topoisomerase II. Finally, TOP-53 displays nearly wild-type activity against a mutant yeast type II enzyme that is highly resistant to etoposide. This finding suggests that TOP-53 can retain activity against systems that have developed resistance to etoposide, and indicates that substituents on the etoposide C-ring are important for topoisomerase II-drug interactions.

DNA abasic lesions in a different light: solution structure of an endogenous topoisomerase II poison.

Topoisomerase II is the target for several anticancer drugs that "poison" the enzyme and convert it to a cellular toxin by increasing topoisomerase II-mediated DNA cleavage. In addition to these "exogenous topoisomerase II poisons," DNA lesions such as abasic sites act as "endogenous poisons" of the enzyme. Drugs and lesions are believed to stimulate DNA scission by altering the structure of the double helix within the cleavage site of the enzyme. However, the structural alterations that enhance cleavage are unknown. Since abasic sites are an intrinsic part of the genetic material, they represent an attractive model to assess DNA distortions that lead to altered topoisomerase II function. Therefore, the structure of a double-stranded dodecamer containing a tetrahydrofuran apurinic lesion at the +2 position of a topoisomerase II DNA cleavage site was determined by NMR spectroscopy. Three major features distinguished the apurinic structure ( = 0.095) from that of wild-type ( = 0.077). First, loss of base stacking at the lesion collapsed the major groove and reduced the distance between the two scissile phosphodiester bonds. Second, the apurinic lesion induced a bend that was centered about the topoisomerase II cleavage site. Third, the base immediately opposite the lesion was extrahelical and relocated to the minor groove. All of these structural alterations have the potential to influence interactions between topoisomerase II and its DNA substrate.

Cytosine arabinoside lesions are position-specific topoisomerase II poisons and stimulate DNA cleavage mediated by the human type II enzymes.

Cytosine arabinoside (araC) is an important drug used for the treatment of human leukemias. In order to exert its cytotoxic effects, araC must be incorporated into chromosomal DNA. Although specific DNA lesions that involve base loss or modification stimulate nucleic acid cleavage mediated by type II topoisomerases, the effects of deoxyribose sugar ring modification on enzyme activity have not been examined. Therefore, the effects of incorporated araC residues on the DNA cleavage/religation equilibrium of human topoisomerase IIalpha and beta were characterized. AraC lesions were position-specific topoisomerase II poisons and stimulated DNA scission mediated by both human type II enzymes. However, the positional specificity of araC residues differed from that previously reported for other cleavage-enhancing DNA lesions. Finally, additive or synergistic increases in DNA cleavage were observed in the presence of araC lesions and etoposide. These findings broaden the range of DNA lesions known to alter topoisomerase II function and raise the possibility that this enzyme may mediate some of the cellular effects of araC.

Azatoxin is a mechanistic hybrid of the topoisomerase II-targeted anticancer drugs etoposide and ellipticine.

One approach to broadening the diversity of topoisomerase II-targeted anticancer agents is to generate novel compounds by combining structural elements of drugs known to stimulate enzyme-mediated DNA cleavage. The first agent to emerge from such a rational drug design is azatoxin, a hybrid drug that fuses chemical structures from etoposide and ellipticine. Since these drugs differ significantly in their structural and mechanistic attributes, azatoxin may preferentially retain the functional properties of one of these two drugs, behave as a hybrid molecule, or act as a novel pharmacophore. Therefore, the properties of azatoxin were characterized to determine relationships between its mechanism of action and those of its parent compounds. Azatoxin, like etoposide, binds to DNA in a nonintercalative fashion. However, similar to ellipticine, the drug has no effect on enzyme-mediated DNA religation and apparently stimulates scission primarily by enhancing cleavage complex formation. Depending on the species of topoisomerase II examined, the cleavage potency of azatoxin resembles that of either of its chemical parents. Furthermore, out of 43 DNA cleavage sites analyzed, approximately 90% of those induced by azatoxin are shared with either etoposide, ellipticine, or both drugs. Finally, competition studies indicate that azatoxin interacts with topoisomerase II in the enzyme domain utilized by etoposide and ellipticine. Taken together, these results strongly suggest that azatoxin is a mechanistic hybrid of its parent compounds and shares functional properties with both drugs.

Elicitation of dihydrobenzophenanthridine oxidase in Sanguinaria canadensis cell cultures.

Dihydrobenzophenanthridine (DHBP) oxidase catalyses the last step in the biogenesis of the benzo[c]phenanthridine alkaloid sanguinarine. Addition of autoclaved fungal preparations or putative plant defence signalling intermediates (jasmonic acid (JA), methyl jasmonate (MeJ), acetylsalicylic acid (ASA)) to Sanguinaria canadensis cell suspension cultures elicited an increase in the activity of DHBP oxidase. MeJ and ASA were better inducers of oxidase activity than were the fungal elicitor and JA. Enzyme-specific activity could be induced in a dose- and time-dependent manner up to 4- to 14-fold, respectively, when cells were treated with MeJ or with ASA. A change in total enzyme activity in cultured cells was observed only at the highest concentration of MeJ and not at any level of ASA tested. The results suggest that MeJ and ASA may play a role in the S. canadensis defence against pathogens by eliciting the enzymes involved in the synthesis of the phytoalexin benzophenanthridine alkaloids.

Structure and mapping of the human thymopoietin (TMPO) gene and relationship of human TMPO beta to rat lamin-associated polypeptide 2.

Thymopoietins (TMPOs, previously abbreviated TPs) alpha (75 kDa), beta (51 kDa), and gamma (39 kDa) are related nuclear proteins expressed in many or all tissues. TMPO alpha is present diffusely throughout the nucleus, while TMPOs beta and gamma are localized to the nuclear membrane. Here we report the cloning and analysis of a single TMPO gene encoding TMPOs alpha, beta, and gamma, which are produced by alternative mRNA splicing, as previously inferred from cDNA sequences. The eight exons of the TMPO gene are spread over approximately 35 kb. Exon 4, which is spliced into TMPO alpha mRNA, contains sequences that encode a putative basic nuclear localization motif. Exon 8, which is spliced into TMPO beta and gamma mRNAs, encodes a hydrophobic putative membrane-spanning domain that is thought to target TMPOs beta and gamma to the nuclear membrane. TMPO beta appears to be the human homologue of the recently described rat protein LAP2 (lamina-associated polypeptide 2), which is thought to play an important role in the regulation of nuclear architecture by binding lamin B1 and chromosomes in a manner regulated by phosphorylation during mitosis (K. Furukawa and L. Gerace, La Jolla, pers. comm., 22 Nov. 1994). The human TMPO gene maps to chromosome band 12q22.

Three distinct human thymopoietins are derived from alternatively spliced mRNAs.

Thymopoietin (TP) was originally isolated as a 5-kDa 49-aa protein from bovine thymus in studies of the effects of thymic extracts on neuromuscular transmission and was subsequently observed to affect T-cell differentiation and function. We now report the isolation of cDNA clones for three alternatively spliced mRNAs that encode three distinct human T-cell TPs. Proteins encoded by these mRNAs, which we have named TP alpha (75 kDa), TP beta (51 kDa), and TP gamma (39 kDa), contain identical N-terminal regions, including sequences nearly identical to that of the originally isolated 49-aa protein, but divergent C-terminal regions. TP mRNAs are expressed in many tissues, most abundantly in adult thymus and fetal liver of the tissues so far examined. Distinct structural domains and functional motifs in TPs alpha, beta, and gamma suggest that the proteins have unique functions and may be directed to distinct subcellular compartments.

Treatment of high-dose intrathecal morphine overdose. Case report.

The case is reported of a 45-year-old woman who was being treated for chronic back and right leg pain with intrathecal morphine administered via a subcutaneous continuous-infusion device. She received an accidental 450-mg bolus injection of morphine intrathecally and developed hypertension, status epilepticus, intracerebral hemorrhage, and respiratory failure. Treatment with continuous intravenous naloxone infusion, lumbar catheter drainage of cerebrospinal fluid, and control of hypertension and status epilepticus resulted in an excellent outcome with return to neurological baseline. Care providers who refill pump reservoirs with morphine must be knowledgeable about these devices and the life-threatening consequences associated with errors in refilling them. This case describes the complications and successful treatment of high-dose intrathecal morphine overdose.

Differential enhancement of benzophenanthridine alkaloid content in cell suspension cultures of Sanguinaria canadensis under conditions of combined hormonal deprivation and fungal elicitation.

An elicitation protocol, resulting in the accumulation of sanguinarine in suspension cultures of Papaver bracteatum, was assessed for induction of the same alkaloid in Sanguinaria canadensis. Although only a trace constituent of P. bracteatum plants, sanguinarine is a major alkaloid (1-3% dry wt) of S. canadensis rhizomes. By combining hormonal deprivation for various intervals and a 3-day fungal (Verticillium dahliae) elicitation, benzophenanthridine alkaloid accumulation was induced in S. canadensis cell suspensions. Chelirubine content increased (0.1-1.3% dry wt) consistently in elicited cell cultures while chelerythrine (0.01-0.10% dry wt) and sanguinarine (0-0.02% dry wt) levels were considerably less. Alkaloid accumulation always occurred upon removal of hormone but only at certain time intervals in the log phase upon fungal elicitation. Levels of dopamine, a precursor of the alkaloids, fluctuated over the incubation period, but displayed a 2- to 6-fold increase in cell suspensions grown without hormone. In some experiments dopamine accumulated to levels > 20% dry wt, and these increases were enhanced by the addition of fungal elicitor. Although the same fungal elicitor induces benzophenanthridines in taxonomically related S. canadensis and P. bracteatum, it did not elicit the accumulation of the same alkaloid in the two different plant cultures.

Transformation of a bop-hop-sop-I-sop-II-Halobacterium halobium mutant to bop+: effects of bacteriorhodopsin photoactivation on cellular proton fluxes and swimming behavior.

We have transformed Pho81, a Halobacterium halobium mutant strain which does not contain any of the four retinylidene proteins known in this species, with the bop gene cluster to create Pho81BR, a BR+HR-SR-I-SR-II-strain. The absorption spectrum, pigment reconstitution process, light-dark adaptation and photochemical reaction cycle of the expressed protein are indistinguishable from those of native bacteriorhodopsin (BR) in purple membrane of wild type strains. Strain Pho81BR permits for the first time characterization of effects of BR photoactivation alone on cell swimming behavior and energetics in the absence of the spectrally similar phototaxis receptor sensory rhodopsin I (SR-I) and electrogenic chloride pump halorhodopsin (HR). A non-adaptive upward shift in spontaneous swimming reversal frequency occurs following 3 s of continuous illumination of Pho81BR cells with green light (550 +/- 20 nm). This effect is abolished by low concentrations of the proton ionophore carbonylcyanide m-chlorophenylhydrazone. Although BR does not mediate phototaxis responses in energized Pho81BR cells under our culture conditions, proton pumping by BR in Pho81BR cells partially deenergized by inhibitors of respiration and adenosine triphosphate synthesis results in a small attractant response. Based on our measurements, we attribute the observed effects of BR photoactivation on swimming behavior to secondary consequences of electrogenic proton pumping on metabolic or signal transduction pathways, rather than to primary sensory signaling such as that mediated by SR-I. Proton extrusion by BR activates gated proton influx ports resulting in net proton uptake in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation of members of the genus Haloarcula with shuttle vectors based on Halobacterium halobium and Haloferax volcanii plasmid replicons.

We have stably transformed both Haloarcula vallismortis and Haloarcula hispanica with the halobacterium-Escherichia coli shuttle vectors pWL102 (based on the Haloferax volcanii pHV2 replicon) and pUBP2 (based on the Halobacterium halobium pHH1 replicon). Haloferax volcanii, Halobacterium halobium, and Haloarcula vailismortis are equally distant from one another and span the phylogenetic depth of the halophilic Archaea; thus, these vectors may be generally useful for the halophiles. Both Haloarcula vallismortis and Haloarcula hispanica exhibit previously unreported complex life cycles and are therefore significant as genetically approachable models of cellular differentiation within the Archaea.

Quantitation of insulin-like growth factor-I binding to highly purified human erythroid colony-forming units.

A method has been developed by which erythroid colony-forming units (CFU-E) may be obtained from human blood in sufficient number and purity for quantitative studies of growth factor binding. Studies in serum-free medium have shown that CFU-E require the addition of only two growth factors, erythropoietin (EP) and insulin-like growth factor-I (IGF-I), for growth and differentiation. The IGF-I may be replaced by higher (100-fold) concentrations of insulin. Incubation of CFU-E with 125I recombinant human IGF-I (rhIGF-I) at 4 degrees C has demonstrated specific binding that is directly proportional to the cell concentration. Competition with unlabeled rhIGF-I markedly decreased binding, whereas other growth factors such as granulocyte-monocyte colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and epidermal growth factor (EGF) had no significant effect on the binding of [125I]rhIGF-I. The binding was saturable at an [125I]rhIGF-I concentration of 10 ng/ml (1.2 nM). Scatchard analysis revealed two classes of IGF-I receptors present on the CFU-E cell surface: a low-affinity class of 549 receptors with Kd = 0.44 nM and a high-affinity class of 341 receptors with Kd = 0.04 nM.

Transformation of the archaebacterium Halobacterium volcanii with genomic DNA.

We describe optimization of a transformation system for the halophilic archaebacterium Halobacterium volcanii. Transformation of spheroplasts in the presence of polyethylene glycol permits the uptake and expression of high-molecular-weight linear fragments of genomic DNA as well as plasmid or bacteriophage DNA. Transformations can be performed with either fresh or frozen cell preparations. Auxotrophic mutants were transformed to prototrophy with genomic DNA from wild-type cells with efficiencies of 5 x 10(4)/micrograms of DNA and frequencies of 8 x 10(-5) per regenerated spheroplast. The overall efficiency of transformation with genomic DNA implies that genetic recombination is an efficient process in H. volcanii.

Transformation methods for halophilic archaebacteria.

We present a practical description of polyethylene glycol mediated spheroplast transformation of Halobacterium halobium and Halobacterium volcanii. This method has been applied to phage DNA transfection, plasmid DNA transformation, and transformation with linear fragments of high molecular weight genomic DNA. Efficient spheroplast regeneration allows uncomplicated recovery of transformed progeny. Transformations can be performed equally well using fresh or frozen cell preparations. These methods should find application in molecular cloning, genetic fine mapping, and strain construction.

Ultrastructural changes associated with the accumulation and secretion of sanguinarine in Papaver bracteatum suspension cultures treated with fungal elicitor.

Suspension cultures of Papaver bracteatum Arya II Lindl., grown without hormone in the presence of conidial extracts of Verticillium dahliae Kleb., accumulate millimolar quantities of the benzophenanthridine alkaloid, sanguinarine. Under the fluorescence microscope, the elicitor-treated cells display an orange-yellow fluorescence characteristic of sanguinarine, primarily near the periphery of the cells. Electron-microscopic inspection showed the presence of slightly dilated endoplasmic reticulum and of electron-dense protuberances on the tonoplast of large central vacuoles. These osmiophilic aggregates lining the tonoplast bud into spherical bodies, appear to become detached from the membrane and are released into the vacuole. Upon subcellular fractionation of elicited cells on Renografin step gradients, sanguinarine was found to be distributed in all bands but with 86% concentrated in the gradient pellet. Analysis of the pellet by electron microscopy showed that it contained electron-dense fragments similar to the osmiophilic bodies observed on the tonoplast of intact elicited cells. In elicited cell cultures, most of the sanguinarine was recovered from medium in a 100·g sedimenting, cell-free, particulate fraction accounting for as much as 85% of the media sanguinarine and 62% of the total sanguinarine. The sanguinarine-rich 100·g media pellet was determined to be two-thirds protein, one-third RNA and was essentially devoid of phenolics, phospholipid and DNA. The pellet consisted of electrondense material and cytoplasmic remnants resembling those found in the Renografin pellet and tonoplast aggregates of intact cells. When placed under hypotonic conditions or extracted with aqueous buffer, pH 3-11, the pellet did not release sanguinarine. These observations provide evidence for storage of sanguinarine at electron-dense deposits which occur on the tonoplast and as freely floating bodies in vacuoles.

Stimulation of Sanguinarine Production by Combined Fungal Elicitation and Hormonal Deprivation in Cell Suspension Cultures of Papaver bracteatum.

Fungal elicitor preparations from either homogenized mycelia of Dendryphion penicillatum (Cda.) Fr., a specific pathogen of Papaver species, or conidia of Verticillium dahliae Kleb., a general pathogen, were added to 14-day-old suspension cultures of Papaver bracteatum. Plant tissue cultures were grown either in the presence or absence of 0.1 milligram of 2,4-dichlorophenoxyacetic acid per liter and 0.5 milligram of 6-benzylam-inopurine per liter. Dendryphion extracts elicited an accumulation of the benzophenanthridine alkaloid, sanguinarine, which was not greatly influenced by hormone deprivation. Millimolar concentrations of dopamine were detected under all conditions. Thebaine was found when cells were cultured in hormone-free media, but it was not elicitor dose dependent. Verticillium-elicited cultures accumulated sanguinarine in an elicitor-dose-dependent manner only under conditions of hormonal deprivation, resulting in an elevation of sanguinarine levels 5- to 500-fold greater than controls (2-10% dry weight). Most of the sanguinarine accumulated in the medium (23 milligrams per liter), with 85% of the alkaloid associated with a 100g sedimenting fraction that, upon light microscopic inspection, proved to be devoid of cells. In bioassays, sanguinarine showed significant biological activity at concentrations as low as 5 to 10 micrograms per milliliter against three general plant pathogens, Verticillium dahliae, Botrytis cinerea Pers. ex Fr., and Rhizoctonia solani Kuehn. Dendryphion was less affected by sanguinarine addition and displayed an ability to metabolize the alkaloid as evidenced by its loss from the media, subsequent accumulation in the mycelia, and ultimate disappearance over a 48-hour period. By comparison, dopamine and thebaine were less toxic to the general plant pathogens.