Genome Engineering of Yarrowia lipolytica with the PiggyBac Transposon System.

A mutant excision/integration piggyBac transposase can be used to seamlessly excise a chromosomally integrated, piggyBac-compatible selection marker cassette from the Yarrowia lipolytica genome. This piggyBac transposase-based genome engineering process allows for both positive selection of targeted homologous recombination events and scarless or footprint-free genome modifications after precise marker recovery. Residual non-native sequences left in the genome after marker excision can be minimized (0-4 nucleotides) or customized (user-defined except for a TTAA tetranucleotide).

T7 Polymerase Expression of Guide RNAs in vivo Allows Exportable CRISPR-Cas9 Editing in Multiple Yeast Hosts.

Efficient guide RNA expression often limits CRISPR-Cas9 implementation in new hosts. To address this limitation in fungal systems, we demonstrate the utility of a T7 polymerase system to effectively express sgRNAs. Initially, we developed a methodology in Saccharomyces cerevisiae using a modified version of the T7 P266L mutant polymerase with an SV40 nuclear localization signal to allow guide RNA expression immediately downstream of a T7 promoter.

Endogenous 5-HT2C Receptors Phosphorylate the cAMP Response Element Binding Protein via Protein Kinase C-Promoted Activation of Extracellular-Regulated Kinases-1/2 in Hypothalamic mHypoA-2/10 Cells.

Serotonin 5-HT2C receptors (5-HT2CR) activate Gq proteins and are expressed in the central nervous system (CNS). 5-HT2CR regulate emotion, feeding, reward, or cognition and may serve as promising drug targets to treat psychiatric disorders or obesity. Owing to technical difficulties in isolating cells from the CNS and the lack of suitable cell lines endogenously expressing 5-HT2CR, our knowledge about this receptor subtype in native environments is rather limited.

Glucose Enhances Basal or Melanocortin-Induced cAMP-Response Element Activity in Hypothalamic Cells.

Melanocyte-stimulating hormone (MSH)-induced activation of the cAMP-response element (CRE) via the CRE-binding protein in hypothalamic cells promotes expression of TRH and thereby restricts food intake and increases energy expenditure. Glucose also induces central anorexigenic effects by acting on hypothalamic neurons, but the underlying mechanisms are not completely understood. It has been proposed that glucose activates the CRE-binding protein-regulated transcriptional coactivator 2 (CRTC-2) in hypothalamic neurons by inhibition of AMP-activated protein kinases (AMPKs), but whether glucose directly affects hypothalamic CRE activity has not yet been shown.

Occurrence of giant focal forms of congenital hyperinsulinism with incorrect visualization by (18) F DOPA-PET/CT scanning.

Context: Congenital hyperinsulinism (CHI) is a rare disease characterized by severe hypoglycaemic episodes due to pathologically increased insulin secretion from the pancreatic beta cells. When untreated, CHI might result in irreversible brain damage and death. Currently, two major subtypes of CHI are known: a focal form, associated with local distribution of affected beta cells and a nonfocal form, affecting every single beta cell.

Novel biological action of the dipeptidylpeptidase-IV inhibitor, sitagliptin, as a glucagon-like peptide-1 secretagogue.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted into the circulation by the intestinal L cell. The dipeptidylpeptidase-IV (DPP-IV) inhibitor, sitagliptin, prevents GLP-1 degradation and is used in the clinic to treat patients with type 2 diabetes mellitus, leading to improved glycated hemoglobin levels. When the effect of sitagliptin on GLP-1 levels was examined in neonatal streptozotocin rats, a model of type 2 diabetes mellitus, a 4.

Mechanisms underlying metformin-induced secretion of glucagon-like peptide-1 from the intestinal L cell.

Glucagon-like peptide-1(7-36NH2) (GLP-1) is secreted by the intestinal L cell in response to both nutrient and neural stimulation, resulting in enhanced glucose-dependent insulin secretion. GLP-1 is therefore an attractive therapeutic for the treatment of type 2 diabetes. The antidiabetic drug, metformin, is known to increase circulating GLP-1 levels, although its mechanism of action is unknown.

Essential role for protein kinase Cζ in oleic acid-induced glucagon-like peptide-1 secretion in vivo in the rat.

Luminal monounsaturated long-chain fatty acids [e.g. oleic acid (OA)] increase secretion of the incretin, glucagon-like peptide-1 (GLP-1) from the ileocolonic L cell.

Carcinogenic effects of exogenous and endogenous glucagon-like peptide-2 in azoxymethane-treated mice.

Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent intestinotropic hormone that promotes intestinal growth, via increased intestinal proliferation and decreased apoptosis, as well as increases in nutrient absorption and barrier function. The long-acting analog h(Gly(2))GLP-2[1-33] is currently being tested for treatment of short bowel syndrome and Crohn's disease. However, the role of GLP-2 in colon carcinogenesis is controversial.

GPR119 is essential for oleoylethanolamide-induced glucagon-like peptide-1 secretion from the intestinal enteroendocrine L-cell.

Objective: Intestinal L-cells secrete the incretin glucagon-like peptide-1 (GLP-1) in response to ingestion of nutrients, especially long-chain fatty acids. The Galphas-coupled receptor GPR119 binds the long-chain fatty acid derivate oleoylethanolamide (OEA), and GPR119 agonists enhance GLP-1 secretion. We therefore hypothesized that OEA stimulates GLP-1 release through a GPR119-dependent mechanism.

Immune restoration in head and neck cancer patients after in vivo COX-2 inhibition.

Purpose: To determine the immunomodulatory effects of in vivo COX-2 inhibition on leukocyte infiltration and function in patients with head and neck cancer.

Experimental Design: Patients with squamous cell carcinoma of the head and neck preoperatively received a specific COX-2 inhibitor (rofecoxib, 25 mg daily) orally for 3 weeks. Serum and tumor specimens were collected at the start of COX-2 inhibition (day 0) and again on the day of surgery (day 21).

Granulomatous Pneumocystis carinii pneumonia in a non-AIDS patient: an atypical presentation.

We present the computed tomographic findings of pulmonary involvement by granulomatous Pneumocystis carinii pneumonia in a 73-year-old woman recently tapered from a high-dose long-term systemic corticosteroid therapy for Factor VII deficiency.

Impaired monocyte function in cancer patients: restoration with a cyclooxygenase-2 inhibitor.

Epidemiological data and animal models have provided evidence that nonsteroidal antiinflammatory drugs (NSAIDs) have an anticancer effect. However, the molecular mechanisms underlying these antineoplastic effects are not well understood. We described previously that expression levels of the chemokine receptor, CCR5, and the beta2-integrin, Mac-1, were down-regulated on primary monocytes after incubation in supernatants from human carcinoma cell lines, and that this down-regulation resulted in impaired monocyte function with respect to migration and adhesion.

Interleukin-1 beta converting enzyme inhibition blocks progression of type II collagen-induced arthritis in mice.

To IL-1 beta is a principal mediator in the pathogenesis of inflammatory disease. The IL-1 beta-converting enzyme (ICE), a novel cysteine protease, is required for processing of the 31 kDa IL-1 beta precursor to generate the 17 kDa proinflammatory mature form. We investigated the effect of two irreversible peptidyl ICE inhibitors, VE-13,045 and VE-16,084, on IL-1 production in vitro and in vivo in acute and chronic inflammatory disease models.

Monomeric and dimeric forms of soluble receptors can differ in their neutralization potential.

Recombinant soluble forms of transmembrane receptors can be produced in monomeric and dimeric versions. Binding affinity and neutralization potential of these different forms of soluble receptors depend on the quaternary structure of their ligands. Monomeric ligands will be bound with equal affinity by both forms, whereas trimeric ligands, e.

N- and O-glycosylation muteins of recombinant human erythropoietin secreted from BHK-21 cells.

Single-site glycomuteins of recombinant human erythropoietin (rhuEpo) were constructed and transiently and stably expressed in BHK-21 cells. The transient expression levels varied among muteins, being highest for mutein rhuEpoGln24 followed by wild-type rhuEpo (rhuEpowt). All other glycomuteins, including rhuEpoGln38, rhuEpoGln83, rhuEpoThr126, and rhuEpoGly126, were secreted at lower levels than rhuEpowt.

The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane.

The signal recognition particle receptor (SR) is required for the cotranslational targeting of both secretory and membrane proteins to the endoplasmic reticulum (ER) membrane. During targeting, the SR interacts with the signal recognition particle (SRP) which is bound to the signal sequence of the nascent protein chain. This interaction catalyzes the GTP-dependent transfer of the nascent chain from SRP to the protein translocation apparatus in the ER membrane.

In vitro and in vivo evaluation of the subtype-selective muscarinic agonist PD 151832.

PD 151832 is a potent partial muscarinic agonist that displays a high level of functional selectivity for the muscarinic m1 receptor subtype, as evidenced by its selective stimulation of PI turnover and cellular metabolic activity in transfected Hm1-CHO cells at concentrations that produce minimal stimulation of other cloned human muscarinic receptors. PD 151832 enhanced the amplification of Hm1-transfected NIH-3T3 cells at concentrations lower than those required to produce similar effects in Hm2 or Hm3-transfected cells. The functional m1 selectivity of PD 151832 is consistent with its improvement of mouse water maze performance at doses far lower than those required to produce peripheral parasympathetic side effects.

Inhibition of allergen-induced IgE and IgG1 production by soluble IL-4 receptor.

In this study, the effect of soluble IL-4 receptors (sIL-4R) on murine allergen-induced IgE and IgG1 production was examined. Lymphocytes from mice sensitized to the allergens ragweed (RW) or ovalbumin (OVA) in vivo were restimulated in vitro with the sensitizing allergen in the presence of either a soluble murine sIL-4R, a dimeric sIL-4R Ig fusion protein (sIL-4R/Fc), or anti-IL-4 antibody in 14-day cultures. Both monomeric and dimeric sIL-4R inhibited polyclonal IgE (approximately 70%) and IgG1 (approximately 35%) production in a dose-dependent fashion, similar to that observed in the presence of the anti-IL-4 antibody.