CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion.

The ability to efficiently produce recombinant proteins in a secreted form is highly desirable and cultured mammalian cells such as CHO cells have become the preferred host as they secrete proteins with human-like post-translational modifications. However, attempts to express high levels of particular proteins in CHO cells may consistently result in low yields, even for non-engineered proteins such as immunoglobulins. In this study, we identified the responsible faulty step at the stage of translational arrest, translocation and early processing for such a "difficult-to-express" immunoglobulin, resulting in improper cleavage of the light chain and its precipitation in an insoluble cellular fraction unable to contribute to immunoglobulin assembly. We further show that proper processing and secretion were restored by over-expressing human signal receptor protein SRP14 and other components of the secretion pathway. This allowed the expression of the difficult-to-express protein to high yields, and it also increased the production of an easy-to-express protein. Our results demonstrate that components of the secretory and processing pathways can be limiting, and that engineering of the secretory pathway may be used to improve the secretion efficiency of therapeutic proteins from CHO cells.

Efeito do tamanho do folículo na maturação nuclear e citoplasmática de ovócitos de fêmeas zebuínas

Este estudo avaliou o efeito do tamanho do folículo na capacidade dos ovócitos de sofrerem maturação nuclear e citoplasmática. Ovários de vacas Nelore (Bos indicus) foram coletados logo após o abate, e os complexos cúmulus-ovócitos foram aspirados de folículos de diferentes categorias, e classificados de acordo com seus diâmetros em: 1-2 mm, 3-5 mm, 6-8 mm e > ou = 9 mm. A medida do diâmetro e a fixação dos ovócitos para avaliação do estádio nuclear antes da maturação foram feitas logo após a aspiração. Ovócitos morfologicamente viáveis foram maturados, fecundados e cultivados in vitro. Os ovócitos obtidos de folículos de 1-2 mm apresentaram menor diâmetro (P<0,01) do que os dos demais grupos. Antes da maturação, 89,8%, 90,1%, 85,7% e 100% dos ovócitos provenientes de folículos de 1-2, 3-5, 6-8 e > ou = 9 mm de diâmetro, respectivamente, apresentavam vesícula germinativa. O tamanho do folículo não influenciou (P<0,05) a taxa de maturação nuclear. A porcentagem de ovócitos que chegaram a metáfase II foi de 88,8%, 87,8%, 92,9% e 100% no que se refere aos ovócitos de folículos de 1-2, 3-5, 6-8 e > ou = 9 mm, respectivamente. As taxas de penetração e clivagem foram semelhantes (P>0,05) nos grupos de 1-2 mm (93,4% e 81,9%), 3-5 mm (90,7% e 79,6%), 6-8 mm (91,3% e 77,8%) e > ou = 9 mm (92,0% e 78,3%). Da mesma forma, não houve diferenças (P>0,05) entre as categorias de folículos no que se refere às taxas de polispermia, descondensação da cabeça do espermatozóide e formação de pro-núcleos. Os resultados deste estudo demonstram que na espécie B. indicus, folículos com diâmetro de 1 mm até > ou = 9 mm não influenciaram a capacidade dos ovócitos de reiniciar e completar a meiose e de clivar após maturação e fecundação in vitro.

Controle da lagarta-da-soja com aplicações de seu vírus de poliedrose nuclear por vias aérea e terrestre

De 1983 a 1988 foram conduzidos, na região de Dourados, MS, seis experimentos e três campos-piloto, objetivando controlar a lagarta Anticarsia gemmatalis Hübner, 1818, com aplicações aérea e terrestre de seu vírus de poliedrose nuclear (VPN Ag). Cem lagartas equivalentes (LE) de VPN Ag associadas a óleo de soja, melaço de cana-de-açúcar e água, foram aplicadas com avião agrícola equipado com Micronair. Os preparados oleosos (5,5 e 5 L ha-1) e com melaço (10 L ha-1) controlaram 75-89% e 79-96% das lagartas, respectivamente. A suspensão aquosa de 3 L ha-1 foi ineficaz, porém as de 15, 20 e 25 L ha-1 controlaram de 81% a 90% das lagartas. Cinqüenta LE, aplicadas com avião agrícola (3 L ha-1) ou atomizador (15 L ha-1), foram ineficientes. Aplicações da mesma dose com pulverizador de barra (134 e 150 L ha-1) proporcionaram controle de 87% e 90%, respectivamente, e com avião (15, 20 e 25 L ha-1), entre 93% e 98%. Aplicações aéreas de 50 LE com óleo de soja (5 L ha-1) ou melaço (10 L ha-1) foram eficientes (86-88% e 99%, respectivamente). Aplicações aéreas de suspensões aquosas e formulado oleoso, em campos-piloto, confirmaram os resultados experimentais.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications.

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Engineering Study for the Evaluation of Public Road Administration and Maintenance Alternatives, Executive Summary for HR-265, 1985

Iowa's public road system of 112,000 miles is one of the largest and the best in the nation. It represents a considerable financial investment of taxpayer revenues over the years. And, it requires a sustained investment to preserve an economical level of transport service into the future. In 1982, a Governor's Blue Ribbon Transportation Task Force evaluated the effectiveness of Iowa's entire transportation system. Four important Task Force recommendations dealt with public road administrative issues in Iowa. These issues were related to: 1. design criteria and levels of maintenance 2. consistency in the use of standards among jurisdictions 3. consolidation of maintenance operations at one jurisdictional level and 4. jurisdictional authority for roads. The issues formed the background for Research Project HR-265.

Field Evaluation of Engineering Fabrics for Asphalt Concrete Resurfacing - Audubon County, HR-360, Construction Report, 1994

An asphalt concrete (ACC) overlay is most often the rehabilitative effort used to maintain the serviceability of either an ACC or PCC pavement. The major problem in durability of this ACC overlay comes from reflective cracking. These cracks usually open, allowing water to enter the unsealed crack and strip the ACC in the overlay. The stripping of the ACC allows accelerated deterioration at the crack. Two engineering fabrics were evaluated in this project in order to determine their effectiveness in reducing reflective cracking. These two materials are: PavePrep, Contech Construction Products, Inc., and Pro-Guard, Phillips Fiber Corporation. A 4.2 km (2.6 mi) roadway in Audubon County was selected for the research project. The roadway was divided into eight test sections. Four of the test sections are conventional resurfacing. The other four sections are split between the two engineering fabrics (two Pro-Guard and two PavePrep). A 75 mm (3 in.) thick overlay was placed over the entire project.

Engineering Fabric Repair Prior to Resurfacing - Roadglas - US 30, HR-525, 1992

In Iowa it is normal procedure to either use partial or full-depth patching to repair deteriorated areas of pavement prior to resurfacing. The Owens/Corning Corporation introduced a repair system to replace the patching process. Their Roadglas repair system was used in this research project on US 30 in Story County. It was installed in 1985 and has been observed annually since that time. There were some construction problems with slippage as the roller crossed the abundant Roadglas binder. It appears the Roadglas system has helped to control reflective cracking in the research areas. Since the time when this project was completed it has been reported that Owens/Corning has discontinued production of the Roadglas system.

An Evaluation of the Troxler 3241-B Nuclear Asphalt Gauge, MLR-85-11, 1986

The Troxler 3241-B Asphalt Content Gauge is intended for rapidly determining the bitumen content of bituminous paving mixtures. A 300 Millicurie Americuium 241: Beryllium source emitts neutrons which are affected by the hydrogen in the mix. The affected neutrons are detected by Helium 3 detectors, counted and computed into a percentage bitumen of the asphalt mix. The current methods of determining the bitumen content of bituminous paving mixtures requires the use of potentially hazardous chemicals and several hours of testing time. When extracted aggregates are not needed, determination of the bitumen content of a paving mixture by the nuclear method may be easier, quicker and potentially safer. The objective of the project is to study the accuracy of the Troxler 3241-B Nuclear Asphalt Content Gauge in measuring the asphalt cement (AC) content of asphalt concrete mixtures produced with different asphalt cements and different aggregates.

Targeting receptor activator of nuclear factor-kappa B as a new therapy for bone metastasis in non-small cell lung cancer.

PURPOSE OF REVIEW: The review aims at comprehensively discussing our current knowledge on bone metastases incidence in non-small cell lung cancer (NSCLC), their related complications as well as clinical impact in patients suffering from advanced disease. RECENT FINDINGS: After evoking the use of zoledronic acid as the established standard of care until recently, the new class of drugs available to prevent skeletal related events and targeting receptor activator of nuclear factor-kappa B (RANK) will be emphasized, reporting on denosumab clinical trials, a RANK-ligand (RANKL) targeting monoclonal antibody. Biological hypothesis regarding their mechanisms of action as well a potential direct impact on tumor cells are described according to the most recent laboratory as well as hypothesis-generating clinical data. SUMMARY: Targeting the RANK pathway is an efficient way to prevent complications of bone metastases in NSCLC. Interesting additional direct effects on tumor biology and evolution are being analyzed and prospectively assessed in clinical trials.

Estrogen-dependent in vitro transcription from the vitellogenin promoter in liver nuclear extracts.

One approach to analyzing the molecular mechanisms of gene expression in vivo is to reconstitute these events in cell-free systems in vitro. Although there is some evidence for tissue-specific transcription in vitro, transcriptionally active extracts that mimic a steroid hormone-dependent enhancement of transcription have not been described. In the study reported here, nuclear extracts of liver from the frog Xenopus laevis were capable of estrogen-dependent induction of a homologous vitellogenin promoter that contained the estrogen-responsive element.

Crystal engineering studies: polymorphs and co-crystals

We review the key topics of one of the areas with the biggest impact of the last years in the chemical and pharmaceutical industry that is Crystal Engineering. The relevance of polymorphs and co-crystals from different points of view is been highlighted and broadly illustrated by means of several recent examples of studies carried out in this field. In addition, the most suitableinstrumental techniques and the intellectual property implications are reviewed.

Nuclear magnetic resonance

This article reviews the principles and methods of nuclear magnetic resonance spectroscopy, and gives examples of applications carried out at ourFacility, which illustrate the capabilities of the technique.

Peroxisome proliferator-activated receptors: a nuclear receptor signaling pathway in lipid physiology.

Peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors that belong to the steroid/thyroid/retinoic acid receptor superfamily. All their characterized target genes encode proteins that participate in lipid homeostasis. The recent finding that antidiabetic thiazolidinediones and adipogenic prostanoids are ligands of one of the PPARs reveals a novel signaling pathway that directly links these compounds to processes involved in glucose homeostasis and lipid metabolism including adipocyte differentiation. A detailed understanding of this pathway could designate PPARs as targets for the development of novel efficient treatments for several metabolic disorders.

Polychlorinated trityl radicals for dynamic nuclear polarization: the role of chlorine nuclei

Polychlorinated trityl radicals bearing carboxylate substituents are water soluble persistent radicals that can be used for dynamic nuclear polarization. In contrast to other trityl radicals, the polarization mechanism differs from the classical solid effect. DFT calculations performed to rationalize this behaviour support the hypothesis that polarization is transferred from the unpaired electron to chlorine nuclei and from these to carbon by spin diffusion. The marked differences observed between neutral and anionic forms of the radical will be discussed.