Chimerization of antibodies by isolation of rearranged genomic variable regions by the polymerase chain reaction

We describe a new method for amplification, by polymerase chain reaction (PCR), of rearranged segments encoding the variable part of light and heavy chains of an antibody (Ab) from the chromosomal DNA of hybridoma cells for the chimerization ofAbs. A fundamental prerequisite for this is the knowledge ofthe exact sequences in the 5’-untranslated region of light and heavy chain mRNA, and of the joining segment used for rearrangement. This allows the design of nondegenerated oligodeoxyribonucleotides for PCR. The primer design permits directional cloning of the amplified, promoterless fragments into cassette vectors, in which they will be linked to the appropriate human constant domains and immunoglobulin (Ig) promoter/enhancer elements. The method is illustrated for chimerization of an Ab directed against the human T-lymphocyte antigen, CD4. The chimerized Ab is secreted in abundant quantities after transfection of the engineered plasmids into non-Ig-producing myeloma cells

Diel pattern of circadian clock and storage protein gene expression in leaves and during seed filling in cowpea (Vigna unguiculata)

Background Cowpea (Vigna unguiculata) is an important source of protein supply for animal and human nutrition. The major storage globulins VICILIN and LEGUMIN (LEG) are synthesized from several genes including LEGA, LEGB, LEGJ and CVC (CONVICILIN). The current hypothesis is that the plant circadian core clock genes are conserved in a wide array of species and that primary metabolism is to a large extent controlled by the plant circadian clock. Our aim was to investigate a possible link between gene expression of storage proteins and the circadian clock. Results We identified cowpea orthologues of the core clock genes VunLHY, VunTOC1, VunGI and VunELF3, the protein storage genes VunLEG, VunLEGJ, and VunCVC as well as nine candidate reference genes used in RT-PCR. ELONGATION FACTOR 1-A (ELF1A) resulted the most suitable reference gene. The clock genes VunELF3, VunGI, VunTOC1 and VunLHY showed a rhythmic expression profile in leaves with a typical evening/night and morning/midday phased expression. The diel patterns were not completely robust and only VungGI and VungELF3 retained a rhythmic pattern under free running conditions of darkness. Under field conditions, rhythmicity and phasing apparently faded during early pod and seed development and was regained in ripening pods for VunTOC1 and VunLHY. Mature seeds showed a rhythmic expression of VunGI resembling leaf tissue under controlled growth chamber conditions. Comparing time windows during developmental stages we found that VunCVC and VunLEG were significantly down regulated during the night in mature pods as compared to intermediate ripe pods, while changes in seeds were non-significant due to high variance. The rhythmic expression under field conditions was lost under growth chamber conditions. Conclusions The core clock gene network is conserved in cowpea leaves showing a robust diel expression pattern except VunELF3 under growth chamber conditions. There appears to be a clock transcriptional reprogramming in pods and seeds compared to leaves. Storage protein deposition may be circadian regulated under field conditions but the strong environmental signals are not met under artificial growth conditions. Diel expression pattern in field conditions may result in better usage of energy for protein storage.This work was supported by the 7th Research Framework Programme of the European Union “Eurolegume (Enhancing of Legumes Growing in Europe through Sustainable Cropping for Protein Supply for Food and Feed)” FP7– 613781. The funding body had no role in the experimental design, analysis or results shown in the manuscript

Reporters of TCR signaling identify arthritogenic T cells in murine and human autoimmune arthritis.

How pathogenic cluster of differentiation 4 (CD4) T cells in rheumatoid arthritis (RA) develop remains poorly understood. We used Nur77-a marker of T cell antigen receptor (TCR) signaling-to identify antigen-activated CD4 T cells in the SKG mouse model of autoimmune arthritis and in patients with RA. Using a fluorescent reporter of Nur77 expression in SKG mice, we found that higher levels of Nur77-eGFP in SKG CD4 T cells marked their autoreactivity, arthritogenic potential, and ability to more readily differentiate into interleukin-17 (IL-17)-producing cells. The T cells with increased autoreactivity, nonetheless had diminished ex vivo inducible TCR signaling, perhaps reflective of adaptive inhibitory mechanisms induced by chronic autoantigen exposure in vivo. The enhanced autoreactivity was associated with up-regulation of IL-6 cytokine signaling machinery, which might be attributable, in part, to a reduced amount of expression of suppressor of cytokine signaling 3 (SOCS3)-a key negative regulator of IL-6 signaling. As a result, the more autoreactive GFPhi CD4 T cells from SKGNur mice were hyperresponsive to IL-6 receptor signaling. Consistent with findings from SKGNur mice, SOCS3 expression was similarly down-regulated in RA synovium. This suggests that despite impaired TCR signaling, autoreactive T cells exposed to chronic antigen stimulation exhibit heightened sensitivity to IL-6, which contributes to the arthritogenicity in SKG mice, and perhaps in patients with RA

Combinatorial functions of two chimeric antibodies directed to human CD4 and one directed to the a-chain of the human interleukin-2 receptor

The general feasibility of chimerization of monoclonal antibodies (mAbs) has already been shown for a large number of them. In order to evaluate in vitro parameters relevant to immunosuppressive therapy, we have chimerized and synthesized two anti-CD4 mAbs recognizing two different epitopes on the human T-lymphocyte antigen, CD4. The chimerized mAbs are produced at levels corresponding to those of the original hybridoma cell lines. With respect to activation of human complement, the individual Abs are negative; however, when used in combination, complement activation was performed. When applied in combination, they were found to modulate the CD4 antigen, whereas the individual mAb do not display this property. Individually they mediate an up to 60% inhibition of the mixed lymphocyte reaction (MLR). However, by combination of an anti-CD4 mAb with one directed against the a-chain of the human IL2 receptor, nearly 100% inhibition of the MLR was achieved, even with reduced dosage of the mAbs. Our data suggest that the combination of an anti-CD4 mAb and an anti-IL2Rcc chain mAb is more effective with respect to immunosuppression than each mAb by itself, indicating that this mAb cocktail could be a new strategy for immunosuppressive therapy

Chimerization of antibodies by isolation of rearranged genomic variable regions by the polymerase chain reaction

We describe a new method for amplification, by polymerase chain reaction (PCR), of rearranged segments encoding the variable part of light and heavy chains of an antibody (Ab) from the chromosomal DNA of hybridoma cells for the chimerization ofAbs. A fundamental prerequisite for this is the knowledge ofthe exact sequences in the 5’-untranslated region of light and heavy chain mRNA, and of the joining segment used for rearrangement. This allows the design of nondegenerated oligodeoxyribonucleotides for PCR. The primer design permits directional cloning of the amplified, promoterless fragments into cassette vectors, in which they will be linked to the appropriate human constant domains and immunoglobulin (Ig) promoter/enhancer elements. The method is illustrated for chimerization of an Ab directed against the human T-lymphocyte antigen, CD4. The chimerized Ab is secreted in abundant quantities after transfection of the engineered plasmids into non-Ig-producing myeloma cells

2009-2010 Master Class - Yael Weiss (Piano)

Mark Kaplan and Yael Weiss in Recital: All Brahms Concert (December 3, 2009) - Programhttps://spiral.lynn.edu/conservatory_masterclasses/1088/thumbnail.jp

ROTULAGEM NUTRICIONAL DE ALIMENTOS E SUA RELAÇÃO COM OS DIREITOS DA PERSONALIDADE E COM OS DIREITOS FUNDAMENTAIS

Sob o enfoque dos direitos da personalidade, o presente artigo, valendo-se do método hipotético-dedutivo, analisa aspectos relacionados à comercialização e consumo de alimentos. Aborda questões específicas quanto à rotulagem destes, considerando que o consumo inadequado (inapropriado ou excessivo, principalmente de ultraprocessados) gera graves consequências para a saúde do ser humano (um dos exemplos mencionados é a obesidade). Analisa, ainda, a atuação da ANVISA quanto às normas que definem as exigências e limites para a rotulagem nutricional, tudo com especial consideração quanto à proteção dos seres humanos, de seus direitos fundamentais e da personalidade (dentre outros, saúde e vida)

O CONSUMO DE ALIMENTOS ULTRAPROCESSADOS E SUA DIVULGAÇÃO, INCLUINDO PUBLICIDADE DIRECIONADA À CRIANÇA: DIREITO BRASILEIRO E DIREITO INTERNACIONAL

O presente artigo examina questões relacionadas ao mercado de alimentos ultraprocessados cujo direcionamento visa o consumo primordialmente por parte de crianças, considerando sua hipervulnerabilidade, seus direitos fundamentais e da personalidade envolvidos. Destaca a questão da oferta desses alimentos (publicidade explícita e implícita), seu consumo pelos menores e consequências das relações de consumo dessa ordem. Sem olvidar legislação internacional à qual faz referência, em nível nacional o trabalho fundamenta-se no conteúdo do sistema de proteção ao consumidor (Lei nº 8.078/90, Lei n° 8.069/90 e normas regulamentares). Baseia-se, para tanto, em pesquisas bibliográficas e vale-se do método hipotético dedutivo para confecção

A INDÚSTRIA DO AGRONEGÓCIO E AS PRÁTICAS DA SUINOCULTURA, À LUZ DA FORÇA NORMATIVA DA PROTEÇÃO DOS ANIMAIS NÃO HUMANOS

O presente artigo tem como objetivo principal analisar as práticas da suinocultura brasileira e os maus-tratos e crueldade exercidos pela atividade. Valendo-se do método hipotético-dedutivo, o trabalho aborda aspectos socioambientais que envolvem a indústria do agronegócio, especialmente o mito de que apenas a carne animal fornece os nutrientes necessários ao corpo humano e a força do agronegócio, atividade com expressiva participação na economia. Traz os fatores capitalistas de produção envolvidos na suinocultura e analisa, ainda, a força normativa que detém a proteção dos animais, à luz das disposições constitucionais constantes dos artigos 170, VI e 225, §1°, VII, CF, que asseguram aos animais proteção contra práticas cruéis e observância, pela ordem econômica, à defesa do meio ambiente enquanto princípio.The main objective of this article is to analyze the practices of Brazilian pig farming and the mistreatment and cruelty exercised by the activity. Using the hypothetical-deductive method, the work addresses socio-environmental aspects that involve the agribusiness industry, especially the myth that only animal meat provides the necessary nutrients to the human body and the strength of agribusiness, an activity with significant participation in the economy. It brings the capitalist factors of production involved in swine farming and also analyzes the normative force that holds the protection of animals, in the light of the constitutional provisions contained in articles 170, VI and 225, §1, VII, CF, which ensure animals protection against cruel practices and observance, by the economic order, to the defense of the environment as a principle