Investigating the effects of Orexin-A on thermogenesis in human deep neck brown adipose tissue.

BACKGROUND: Despite successful preclinical testing, 85% of early clinical trials for novel drugs fail. Most futilities originate from molecular mechanisms of the drug(s) tested. It is critically important to develop validated human cell-based model systems in which animal-based research can be translated in order to complement the preclinical in vivo findings prior to implementation of a clinical trial. Obesity is associated with reduced circulating levels of Orexin-A (OX-A) in humans. OX-A increases thermogenesis in rodent brown adipose tissue (AT), yet this phenomenon has not been explored in humans. METHODS: We established a cell-based model system of human brown and white adipocytes and tested the effects of OX-A on thermogenesis. RESULTS: Contrary to published in vivo and in vitro reports in rodents, OX-A treatment alone or in combination with an adrenergic stimulus did neither enhance thermogenesis nor its related transcriptional program in a human in vitro model of brown adipocytes or AT explants. CONCLUSIONS: Translating preclinical findings in human model systems poses a challenge that must be overcome for the development of effective therapeutic compounds and targets.

Combination Regimens for Treatment of Carbapenem-Resistant Klebsiella pneumoniae Bloodstream Infections.

Previous studies reported decreased mortality in patients with carbapenemase-producing Klebsiella pneumoniae bloodstream infections (BSIs) treated with combination therapy but included carbapenem-susceptible and -intermediate isolates, as per revised CLSI breakpoints. Here, we assessed outcomes in patients with BSIs caused by phenotypically carbapenem-resistant K. pneumoniae (CRKP) according to the number of in vitro active agents received and whether an extended-spectrum beta-lactam (BL) antibiotic, including meropenem, or an extended-spectrum cephalosporin was administered. We retrospectively reviewed CRKP BSIs at two New York City hospitals from 2006 to 2013, where all isolates had meropenem or imipenem MICs of ≥4 µg/ml. Univariate and multivariable models were created to identify factors associated with mortality. Of 141 CRKP BSI episodes, 23% were treated with a single active agent (SAA), 26% were treated with an SAA plus BL, 28% were treated with multiple active agents (MAA), and 23% were treated with MAA plus BL. Ninety percent of isolates had meropenem MICs of ≥16 µg/ml. Thirty-day mortality was 33% overall and did not significantly differ across the four treatment groups in a multivariable model (P = 0.4); mortality was significantly associated with a Pitt bacteremia score of ≥4 (odds ratio [OR], 7.7; 95% confidence interval [CI], 3.2 to 18.1; P = 0.1), and immunosuppression was protective (OR, 0.4; 95% CI, 0.2 to 1.0; P = 0.04). Individual treatment characteristics were also not significantly associated with outcome, including use of SAAs versus MAA (26% versus 38%, P = 0.1) or BL versus no BL (26% versus 39%, P = 0.1). In summary, in patients with CRKP BSIs caused by isolates with high carbapenem MICs, the role of combination therapy remains unclear, highlighting the need for prospective studies to identify optimal treatment regimens.

The chemotherapeutic agent paclitaxel inhibits autophagy through two distinct mechanisms that regulate apoptosis.

Anti-mitotic agents such as paclitaxel and docetaxel are widely used for the treatment of breast, ovarian and lung cancers. Although paclitaxel induces apoptosis, this drug also modulates autophagy. How autophagy affects paclitaxel activity, is unclear. We discovered that paclitaxel inhibited autophagy through two distinct mechanisms dependent on cell cycle stage. In mitotic cells, paclitaxel blocked activation of the class III phosphatidyl inositol 3 kinase, Vps34, a critical initiator of autophagosome formation. In non-mitotic paclitaxel-treated cells, autophagosomes were generated but their movement and maturation was inhibited. Chemically or genetically blocking autophagosome formation diminished paclitaxel-induced cell death suggesting that autophagosome accumulation sensitized cells to paclitaxel toxicity. In line with these observations, we identified that primary breast tumors that expressed diminished levels of autophagy-initiating genes were resistant to taxane therapy, identifying possible mechanisms and prognostic markers of clinical chemotherapeutic resistance.

The platelet-surface thiol isomerase enzyme ERp57 modulates platelet function.

BACKGROUND: Thiol isomerases are a family of endoplasmic reticulum enzymes which orchestrate redox-based modifications of protein disulphide bonds. Previous studies have identified important roles for the thiol isomerases PDI and ERp5 in the regulation of normal platelet function. AIM: Recently, we demonstrated the presence of a further five thiol isomerases at the platelet surface. In this report we aim to report the role of one of these enzymes - ERp57 in the regulation of platelet function. METHODS/RESULTS: Using enzyme activity function blocking antibodies, we demonstrate a role for ERp57 in platelet aggregation, dense granule secretion, fibrinogen binding, calcium mobilisation and thrombus formation under arterial conditions. In addition to the effects of ERp57 on isolated platelets, we observe the presence of ERp57 in the developing thrombus in vivo. Furthermore the inhibition of ERp57 function was found to reduce laser-injury induced arterial thrombus formation in a murine model of thrombosis. CONCLUSIONS: These data suggest that ERp57 is important for normal platelet function and opens up the possibility that the regulation of platelet function by a range of cell surface thiol isomerases may represent a broad paradigm for the regulation of haemostasis and thrombosis.

Alterations of uromodulin biology: a common denominator of the genetically heterogeneous FJHN/MCKD syndrome.

Autosomal dominant hyperuricemia, gout, renal cysts, and progressive renal insufficiency are hallmarks of a disease complex comprising familial juvenile hyperuricemic nephropathy and medullary cystic kidney diseases type 1 and type 2. In some families the disease is associated with mutations of the gene coding for uromodulin, but the link between the genetic heterogeneity and mechanism(s) leading to the common phenotype symptoms is not clear. In 19 families, we investigated relevant biochemical parameters, performed linkage analysis to known disease loci, sequenced uromodulin gene, expressed and characterized mutant uromodulin proteins, and performed immunohistochemical and electronoptical investigation in kidney tissues. We proved genetic heterogeneity of the disease. Uromodulin mutations were identified in six families. Expressed, mutant proteins showed distinct glycosylation patterns, impaired intracellular trafficking, and decreased ability to be exposed on the plasma membrane, which corresponded with the observations in the patient's kidney tissue. We found a reduction in urinary uromodulin excretion as a common feature shared by almost all of the families. This was associated with case-specific differences in the uromodulin immunohistochemical staining patterns in kidney. Our results suggest that various genetic defects interfere with uromodulin biology, which could lead to the development of the common disease phenotype. 'Uromodulin-associated kidney diseases' may be thus a more appropriate term for this syndrome.

Apical localization of wingless transcripts is required for wingless signaling.

Many developing and adult tissues are comprised of polarized epithelia. Proteins that are asymmetrically distributed in these cells are thought to be localized by protein trafficking. Here we show that the distribution and function of the signaling protein Wingless is predetermined by the subcellular localization of its mRNA. High-resolution in situ hybridization reveals apical transcript localization in the majority of tissues examined. This localization is mediated by two independently acting elements in the 3' UTR. Replacement of these elements with non- or basolaterally localizing elements yields proteins with altered intracellular and extracellular distributions and reduced signaling activities. This novel aspect of the wingless signaling pathway is conserved and may prove to be a mechanism used commonly for establishing epithelial cell polarity.

FTZ-Factor1 and Fushi tarazu interact via conserved nuclear receptor and coactivator motifs.

To activate transcription, most nuclear receptor proteins require coactivators that bind to their ligand-binding domains (LBDs). The Drosophila FTZ-Factor1 (FTZ-F1) protein is a conserved member of the nuclear receptor superfamily, but was previously thought to lack an AF2 motif, a motif that is required for ligand and coactivator binding. Here we show that FTZ-F1 does have an AF2 motif and that it is required to bind a coactivator, the homeodomain-containing protein Fushi tarazu (FTZ). We also show that FTZ contains an AF2-interacting nuclear receptor box, the first to be found in a homeodomain protein. Both interaction motifs are shown to be necessary for physical interactions in vitro and for functional interactions in developing embryos. These unexpected findings have important implications for the conserved homologs of the two proteins.

Distinct inhibitory effects of 2-chloro-2'-deoxyadenosine and 9-beta-D-arabinosyl-2-fluoroadenine on DNA methyltransferase in human T-lymphocytes.

The effect of 2-chloro-2'-deoxyadenosine and 9-beta-D-arabinosyl-2-fluoroadenine on DNA methyltransferase activity in stimulated human T-lymphocytes was estimated. In comparative studies 5-aza-deoxycytidine and deoxyadenosine plus deoxycoformycin were used. These antileukemic compounds demonstrated different effects; both 2CdA and dAdo plus dCF, like 5-aza-dCyt, inhibited the enzyme activity by 85-90% after 72 hours activation of lymphocytes, while the effect of F-ara-A, under the same conditions, was insignificant.

Molecular interactions between Vestigial and Scalloped promote wing formation in Drosophila.

Scalloped (Sd) and Vestigial (Vg) are each needed for Drosophila wing development. We show that Sd is required for Vg function and that altering their relative cellular levels inhibits wing formation. In vitro, Vg binds directly to both Sd and its human homolog, Transcription Enhancer Factor-1. The interaction domains map to a small region of Vg that is essential for Vg-mediated gene activation and to the carboxy-terminal half of Sd. Our observations indicate that Vg and Sd function coordinately to control the expression of genes required for wing development, which implies that Vg is a tissue-specific transcriptional intermediary factor of Sd.

A genetic and molecular analysis of an invectedDominant mutation in Drosophila melanogaster.

The invected gene of Drosophila melanogaster is a homeobox-containing gene that is closely related to engrailed. A dominant gain of function allele, invectedDominant, was derived from mutagenesis of a dominant allele of vestigial, In(2R)vgW. A careful analysis of the phenotype of invectedDominant shows that it is associated with a transformation of the anterior compartment of the wing to a posterior fate. This transformation is normally limited to the wing blade itself and does not involve the remaining tissues derived from the wing imaginal disc, including the wing hinge and dorsal thorax of the fly. The ectopic expression of invected protein associated with invectedDominant correlates spatially with the normal expression pattern of vestigial in the wing imaginal disc, suggesting that control elements of vestigial are driving ectopic invected expression. This was confirmed by sequence analysis that shows that the dominant vestigial activity was eliminated by a deletion that removes the 3' portion of the vestigial coding region. This leaves a gene fusion wherein the vestigial enhancer elements are still juxtaposed immediately 5' to the invected transcriptional start site, but with the vg sequences harboring an additional lesion. Unlike recessive invected alleles, the invectedDominant allele produces an observable phenotype, and as such should prove useful in determining the role of invected in patterning the wing imaginal disc. Genetic analysis has shown that mutations of polyhomeotic, a gene involved in regulating engrailed expression, cause a reproducible alteration in the invectedDominant phenotype. Finally, the invectedDominant allele should prove valuable for identifying and characterizing genes that are activated within the posterior compartment. A screen using various lacZ lines that are asymmetrically expressed in an anterior-posterior manner in the wing imaginal disc isolated one line that shows posterior-specific expression within the transformed anterior compartment.

Use of 5-nitroindole-2'-deoxyribose-5'-triphosphate for labelling and detection of oligonucleotides.

The 5'-triphosphate of 5-nitroindole-2'-deoxyriboside has been shown to be a good substrate for terminal deoxynucleotidyl transferase (TdT). An antibody has been prepared for the detection of 5-nitroindole and has been used for the detection of 5-nitroindole tailed DNA both in single-stranded form and after hybridisation to a template. This is therefore a new method for the detection of nucleic acid probes.

The effect of dominant vestigial alleles upon vestigial-mediated wing patterning during development of Drosophila melanogaster.

The vestigial gene product is required for the completion of wing development in Drosophila melanogaster. In the absence of vestigial gene expression, cells within the larval wing and haltere imaginal discs fail to proliferate normally thus producing adults with severely reduced wings. Of a large number of vestigial mutations that have been characterized, only two are currently known to exist, vestigial(U) and vestigial(W), which manifest a significant dominant phenotype. Both are associated with chromosomal inversions that fuse the majority of the vestigial coding regions to other genes; mastermind in vestigial(U) and invected in vestigial(W) Examination of vestigial expression in the presence of these dominant alleles shows alterations in the disc-specific expression of vestigial during later stages of larval development. These patterning disruptions are specific to cells of the wing imaginal disc, as significant suppression of total levels of vestigial expression within entire larvae could not be detected. This dominant interference of vestigial patterning appears to be mediated in part by the vestigial coding sequences that are within the gene fusions. Further evidence that the dominant phenotype is the result of disrupted vestigial patterning comes from observations that the dominant alleles can be partially suppressed by mutations within the Drosophila-epidermal growth factor receptor gene. Mutagenesis of vestigial(U) and vestigial(W) produced a series of alleles with partially dominant phenotypes that restored various amounts of the adult wing. These phenotypes can be correlated with alterations in specific portions of the vestigial sequences associated with the dominant alleles. In the presence of these partially dominant alleles, wing imaginal discs have significantly more cells which express vestigial compared with the number associated with the original dominant phenotype. Additionally, eliminating some of the dominant effect causes alterations in the patterns of early stage apoptotic cell death associated with dominant vestigial alleles. Utilizing these new vestigial alleles, it is possible to correlate the consequence of altered vestigial expression to subsequent changes in patterning of the wing disc.

The influence of 2-chloro-2'-deoxyadenosine on metabolism of deoxyadenosine in human primary CNS lymphoma.

The effects of 2-chloro-2'-deoxyadenosine (2CdA) on the activity of enzymes important for the metabolism of deoxyadenosine were studied in lysates prepared from human primary central nervous system (CNS) lymphomas and normal human lymphocytes. Strong inhibition (approximately 100%) of the phosphorylation of deoxyadenosine to its deoxynucleotide phosphate derivatives was produced in both systems in the presence of 2CdA, which was phosphorylated concomitantly to 2-chloro-2'-deoxyAMP. Interestingly, 2CdA was also found to be an inhibitor of the deamination of both deoxyadenosine (over 50%) and AMP (70%). These findings add to our understanding of the mechanisms of toxicity of this drug, especially considering that 2CdA is resistant to deamination by adenosine deaminase. These results challenge the existing theories of 2CdA toxicity, which have been limited to the formation of phosphate derivatives of 2CdA. The present in vitro studies have demonstrated that 2CdA also inhibits both phosphorylation and deamination of deoxyadenosine (dAdo), suggesting that its mechanism of toxicity includes a block in dAdo metabolic pathways. This has important implications for the perturbation of cell methylation, a functionality associated with, for example, apoptosis.

Distinguishable functions for engrailed and invected in anterior-posterior patterning in the Drosophila wing.

Subdivision of the limb primordia into compartments initiates pattern formation in the developing limbs. Interaction between distinctly specific cells in adjacent compartments leads to localized expression of the secreted signalling molecules Wingless (Wg) or Decapentaplegic (Dpp), which in turn organize pattern and control growth of the limbs. The homeobox gene engrailed has been implicated in specification of posterior cell fate, whereas the LIM/homeobox gene, apterous, specifies dorsal fate. Removing apterous activity causes a complete transformation from dorsal to ventral fate and leads to the formation of an ectopic dorsal-ventral boundary organizer. By contrast, removing engrailed activity causes incomplete morphological transformation from posterior to anterior fate in the wing, and fails to produce an ectopic anterior-posterior organizer (reviewed in ref.2). Complete transformation can only be effected by simultaneously eliminating activity of engrailed and its homologue invected. Here we show that invected functions principally to specify posterior cell fate. Thus establishment of the anterior-posterior organizer and control of compartment identity are genetically distinguishable, and invected may perform a discrete subset of functions previously ascribed to engrailed.

The chaplain's role in bioethical decision-making.

With advances in medical technology, difficult questions of "What should be done?" and "Who should decide?" are a daily occurrence in hospitals. This paper reports the results of a survey of Canadian chaplains with respect to their involvement in bioethical decision-making. The survey suggests that chaplains make a significant contribution to discussion and resolution of bioethical dilemmas. Using a case study, the paper elaborates on the chaplain's role in bioethical decision-making, and indicates how such participation can influence both cost containment and risk management.

Substrate specificity of methylthioadenosine phosphorylase from human liver.

Methylthioadenosine (MTA) phosphorylase purified 615-fold from human liver cleaved phosphorolytically nucleoside analogues at the decreasing specific activity: 5'-deoxyadenosine > 5'-iodo-5'-deoxyadenosine > MTA > adenosine > 2-chloroadenosine > 2-chloro-5'-O-methyl-2'-deoxyadenosine > 2-chloro-2'-deoxyadenosine > > 2'-deoxyadenosine. Adenosine and analogues of 5'-deoxyadenosine were strong competitive inhibitors of MTA phosphorolysis catalysed by the human liver enzyme.

Dopamine modulation of prolactin and vasopressin but not behavior on satiation of sheep.

We have investigated the rapid changes in plasma prolactin and arginine vasopressin (AVP) associated with water satiation in ruminants. Sheep deprived of water for 72 h were allowed voluntary access to ad libitum water. Each sheep drank approximately 5 liters in a single draft. This was performed in a head-down position, which was maintained for approximately 2-3 min and ceased rapidly with an abrupt movement to the upright position. During dehydration, plasma sodium, osmolality, and AVP all increased significantly, but prolactin was unaffected. On rehydration, AVP fell to basal levels within 5 min, but prolactin increased as a short pulse, after which it rapidly fell again. Plasma sodium and osmolality returned to predehydration levels within 6 h of satiation. A possible role of dopaminergic mechanisms in these responses was investigated by the administration of the dopamine agonist bromocriptine or the antagonist metoclopramide. Neither of these agents had any observable effects on the drinking behavior of the sheep during water satiation. During metoclopramide treatment, dehydration was associated with a marked fall in prolactin, and on rehydration there was a prompt and very marked increase in prolactin level. There was also an exaggerated increase of AVP during dehydration and a decrease on rehydration. Bromocriptine had relatively little effect on prolactin responses but prevented the drinking-associated inhibition of AVP, demonstrating an unexpected dissociation between AVP secretion and the stereotyped drinking response.

Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells.

Five esters of luciferin were synthesized and compared with native luciferin as substrates for firefly luciferase expressed in live intact mammalian cells. The esters themselves were not substrates for purified luciferase, but four were substrates for a purified esterase and all appeared to be hydrolysed to luciferin within mammalian cells. At a substrate concentration of 0.01 mM, the peak luminescence from the cos cells expressing luciferase was up to 6-fold greater with the esters than with unmodified luciferin. At 0.1 mM, the difference between luciferin and the esters was decreased. The kinetics of the luminescent signal with the different luciferin esters varied significantly, indicating possible differences in the rates of uptake, breakdown and enzyme inhibition. The esters did not support luminescence from Escherichia coli cells expressing firefly luciferase, suggesting a lack of appropriate esterase activity in this particular strain. The esters could be useful for the assay of luciferase expression in intact mammalian cells when luciferin levels are limiting, for example in tissues, and in plants. Alternative luciferin derivatives may allow further improvements in sensitivity.