The Elucidation of the involvement of endonuclease DNase y in reducing DNA transfection efficiency in mammalian cells

Gene therapy is predicated upon efficient gene transfer. While viral vectors are the method of choice for transformation efficiency, the immunogenicity and safety concerns remain problematic. Non-viral vectors, on the other hand, have shown high degrees of safety and are mostly non-immunogenic in nature. However, non-viral vectors usually suffer from low levels oftransformation efficiency and transgene expression. Thus, increasing transformation efficiency ofnon-viral vectors, in particular by calcium phosphate co-precipitation technique, is a way of generating a suitable vector for gene therapy and is the aim of this study. It is a long known fact that different cell lines have different transfection efficiencies regardless oftransfection methodology (Lin et a!., 1994). Using commonly available cell lines Madine-Darby Bovine Kidney (MDBK), HeLa and Human Embryonic Kidney (HEK-293), we have shown a decreasing trend ofDNase activity based on a plasmid digestion assay. From densitometry studies, as much as a 40% reduction in DNase activity was observed when comparing HEK-293 (least active) to MDBK (most active). Using various biochemical assays, it was determined that DNase y, in particular, was expressed more highly in MDBK cells than both HeLa and HEK-293. Upon cloning of the bovine DNase y gene, we utilized the sequence information to construct antisense expressing plasmids via both traditional antisense RNA (pASDGneoM) and siRNA (psiRNA-S4, psiRNA-S11 and psiRNA-S16). For the construction ofpASDGneoM, the 3' end of the DNase y was inserted in opposite orientation under a cytomegalovirus (CMV) promoter such that the expression ofRNA complementary to the DNase 2 ymRNA occurred. For siRNA plasmids, the sequence was screened to yield optimal short sequences for siRNA inhibition. The silencing ofbovine DNase y led to an increase in transfection efficiency based on traditional calcium phosphate co-precipitation technique; stable clones of siRNA-producing MDBK cell lines (psiRNA-S4 Bland psiRNA-S4 B4) both demol).strated 4-fold increases in transfection efficiency. Furthermore, serial transfection of antisense DNase y plasmid pASDGneoM and reporter pCMV-~ showed a maximum of 8-fold increase in transfection efficiency when the two separate transfections were carried out 4 hours apart (i.e. transfection ofpASDGneoM, separated by four hours, then transfection ofpCMV-~). Together, these results demonstrate the involvement ofDNase y in reducing transfection efficiency, at least by traditional calcium phosphate technique.

Letter from the Chairman of the Committee of Ways and Means to the Secretary of the Treasurer on the subject of a system of revenue to revive and maintain unimpaired the public credit, with the answer of the Secretary thereto. (1814)

October 18, 1814. Read, and committed to the Committee of the whole House on the report of the Committee of Ways and Means on so much of the President's message as relates to the finances of the United States.

Letter with the letterhead “H.H. Warner and Co.” of Rochester, N.Y. from the H.H. Warner Co

Letter with the letterhead “H.H. Warner and Co.” of Rochester, N.Y. from the H.H. Warner Co. The writer asks why Mr. Woodruff did not stop there on his way home from Pennsylvania regarding his shooting share at Point Mouillee [a game area in Monroe County, Michigan]. This letter is accompanied by an envelope, May 19, 1886.

Receipt from Brush Brothers of Buffalo, N.Y.

Receipt from Brush Brothers of Buffalo, N.Y. for brick, Oct. 26, 1875.

The battle of Buena Vista ; with the operations of the Army of occupation for one month

UANL

Enhancement Of High Affinity Y-Aminobutyric Acid Receptor Binding In Cerebellum Of Pyridoxine-Deficient Rat

The high-affinity of [3H]y-aminobutyric acid (GABA) to GABAA receptors and [3H]baclofen to GABAB receptors were studied in the cerebellum of pyridoxine-deficient rats and compared to pyridoxine-supplemented controls. There was a significant increase in the maximal binding ( Bmax) of both GABAA and GABAB receptors with no significant difference in their binding affinities (Kd). The changes observed suggest a supersensitivity of GABAA and GABAB receptors which seems to correlate negatively with the concentration of GABA in the cerebellum of pyridoxine-deficient rats.

Educar al nuevo soberano : Chile entre 1810 y 1814

Resumen tomado de la publicaci??n

Locus of control, estabilidad y controlabilidad como factores cognitivos de los logros académicos.

Estudiar la relación existente entre la atribución de causas, tipo cognitivo motivacional, y el rendimiento académico en sujetos universitarios. 172 alumnos de cuarto de Psicología y sexto de Telecomunicaciones. Aplicación de las pruebas: escala de controlabilidad, BLS-IV (test de inteligencia), EPQ-A (test de personalidad), y escala de internalidad/externalidad. Éstas fueron administradas por tres psicólogos, en una única sesión que duró aproximadamente hora y media. La corrección de las pruebas se realizó siguiendo las instrucciones que figuraban en los manuales de los tests. Las variables dependientes son las puntuaciones obtenidas y el rendimiento académico. Las variables independientes son: tipo cognitivo-motivacional, personalidad e inteligencia, tipo de estudios, sexo, centro de estudios. La escala de internalidad/externalidad de Lavenson y la de controlabilidad de Gregory, a priori semejantes, han presentado diferente comportamiento en la clasificación de sujetos según su tipo cognitivo-motivacional. No existen diferencias significativas en el rendimiento académico en función del tipo cognitivo motivacional. A igual tipo cognitivo-motivacional siendo éste de externos defensivos, las diferencias en rendimiento académico surgen entre centros de estudios, mientras que considerando los grupos de internos, externos y externos éxito, las diferencias surgen entre sexos.

Refutations and the Logic of Practice. 'Refutaciones y la Lógica de la Práctica'.

Resumen basado en el de la publicación

A battle of elephants: (I)legitimate ethical voices?

In this paper, we aim to distil myriad stakeholder voices through a Foucaudian theoretical apparatus in the establishment of ethical stakeholder voices amidst a takeover of a Ghanaian National Bank with ownership control by the state National Pension Fund and Insurance Company. Resonating with Foucault’s position that, the prove and an actual practice of ethical principles despite risk is non-existent within a democracy, this paper reveals how stakeholders in a takeover further their own interest to the neglect of the very germane societal expectation of a salient stakeholder role. We further fill an existing gap within the stakeholder literature that posit of stakeholders as always possessing the right and ethical voices. We conclude that, despite Foucault’s last lecture of The Courage of Truth: The Government of the Self and others having proven of a robust apparatus in distilling ethical voices from non-ethical within the realm of a democratic field, its idealist nature demands a counterfactual position.

Effects of strontium and calcium simultaneous substitution on electrical and structural properties of Pb1-x-y Ca (x) Sr (y) TiO3 thin films

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sex chromosome evolution in fish: the formation of the neo-Y chromosome in Eigenmannia (Gymnotiformes)

Chromosomes of a species of Eigenmannia presenting a X1X1X2X2:X1X2Y sex chromosome system, resulting from a Y-autosome Robertsonian translocation, were analyzed using the C-banding technique, chromomycin A(3) (CMA(3)) and mithramycin (MM) staining and in situ digestion by the restriction endonuclease AluI. A comparison of the metacentric Y chromosome of males with the corresponding acrocentrics in females indicated that a C-band-positive, CMA(3)/MM-fluorescent and AluI digestion-resistant region had been lost during the process of translocation, resulting in a diminution of heterochromatin in the males. It is hypothesized that the presence of a smaller amount of G+C-rich heterochromatin in the sex chromosomes of the heteromorphic sex when compared with the homomorphic sex may be associated with the sex determination mechanism in this species and may be a more widely occurring phenomenon in fish with differentiated sex chromosomes than was initially thought.