1,721,039 research outputs found
The use of the RACE method to clone hybridoma cDNA when V region primers fail
No full textThe technique of V region PCR to clone antibody V regions from hybridomas has been extensively used. However, in addition to, or even instead of, cloning the V regions with the desired specificity, myeloma cell derived V regions, V regions which are the result of non-productive rearrangements, and also V regions which are productive but which do not recognise the antigen of interest, may be isolated. In this paper we describe a comparison of the use of V region PCR and a modification of the RACE technique to clone the V region of the anti-NGF hybridoma, alpha D11. This hybridoma has heavy and light chain V regions which are refractory to amplification with V region primers, but which are easily amplified using RACE, a PCR based procedure which is independent of the variability within the V regions
Cloning and expression of an anti-nerve growth factor (NGF) antibody for studies using the neuroantibody approach
No full text1. The neuroantibody approach, based on the expression of selected monoclonal antibodies by cells of the nervous system, has recently been described (Cattaneo and Neuberger, 1987; Piccioli et al., 1991). In order to apply this experimental strategy to study the role of nerve growth factor (NGF) in the central nervous system (CNS), we exploited the monoclonal antibody (mAb), alpha D11, which neutralizes very efficiently the biological activity of NGF, both in vitro and in vivo (Cattaneo et al., 1988). 2. The alpha D11 antibody chains were cloned and expressed in COS cells as rat/human chimaeric proteins. The cloned antibody was shown to display all the properties of the parental alpha D11 antibody, including its ability to neutralize NGF biological activity. 3. This will allow us to engineer the expression of recombinant alpha D11 antibodies in the CNS, to study the role of NGF in the developing and adult nervous system. This approach can be extended to other neurotrophic factors for which neutralizing monoclonal antibodies are available
NGF and the Amyloid Precursor Protein in Alzheimer’s Disease: From Molecular Players to Neuronal Circuits
No full textAlzheimer's disease (AD), one of the most common causes of dementia in elderly people, is characterized by progressive impairment in cognitive function, early degeneration of basal forebrain cholinergic neurons (BFCNs), abnormal metabolism of the amyloid precursor protein (APP), amyloid beta-peptide (A beta) depositions, and neurofibrillary tangles. According to the cholinergic hypothesis, dysfunction of acetylcholine-containing neurons in the basal forebrain contributes markedly to the cognitive decline observed in AD. In addition, the neurotrophic factor hypothesis posits that the loss nerve growth factor (NGF) signalling in AD may account for the vulnerability to atrophy of BFCNs and consequent impairment of cholinergic functions. Though acetylcholinesterase inhibitors provide only partial and symptomatic relief to AD patients, emerging data from in vivo magnetic resonance imaging (MRI) and positron emission tomography (PET) studies in mild cognitive impairment (MCI) and AD patients highlight the early involvement of BFCNs in MCI and the early phase of AD. These data support the cholinergic and neurotrophic hypotheses of AD and suggest new targets for AD therapy.Different mechanisms account for selective vulnerability of BFCNs to AD pathology, with regard to altered metabolism of APP and tau. In this review, we provide a general overview of the current knowledge of NGF and APP interplay, focusing on the role of APP in regulating NGF receptors trafficking/signalling and on the involvement of NGF in modulating phosphorylation of APP, which in turn controls APP intracellular trafficking and processing. Moreover, we highlight the consequences of APP interaction with p75NTR and TrkA receptor, which share the same binding site within the APP juxtamembrane domain. We underline the importance of insulin dysmetabolism in AD pathology, in the light of our recent data showing that overlapping intracellular signalling pathways stimulated by NGF or insulin can be compensatory. In particular, NGF-based signalling is able to ameliorates deficiencies in insulin signalling in the medial septum of 3xTg-AD mice. Finally, we present an overview of NGF-regulated microRNAs (miRNAs). These small non-coding RNAs are involved in post-transcriptional regulation of gene expression, and we focus on a subset that are specifically deregulated in AD and thus potentially contribute to its pathology
A critical period in the sensitivity of basal forebrain cholinergic neurones to NGF deprivation
No full textCholinergic neurones of basal forebrain (BF) express receptors for nerve growth factor (NGF) and are sensitive to NGF. In many CNS structures, including the BF region, the expression of NGF and its receptors is developmentally regulated. To test whether BF neurones depend on NGF during restricted time windows of postnatal development, we antagonized endogenous NGF by implanting, in the lateral ventricle of the rat, hybridoma cells producing blocking antibodies specific for NGF. Implants were performed at postnatal day 2 (P2), P8 and P15. BF cholinergic neurones were drastically reduced in number only when endogenous NGF was antagonized during the first postnatal week. We conclude that BF cholinergic neurones are sensitive to NGF deprivation only during early postnatal development
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