Multiphoton ANS fluorescence microscopy as an in vivo sensor for protein misfolding stress.
ABSTRACT The inability of cells to maintain protein folding homeostasis is implicated in the development of neurodegenerative diseases, malignant transformation, and aging. We find that multiphoton fluorescence imaging of 1-anilinonaphthalene-8-sulfonate (ANS) can be used to assess cellular responses to protein misfolding stresses. ANS is relatively nontoxic and enters live cells and cells or tissues fixed in formalin. In an animal model of Alzheimer's disease, ANS fluorescence imaging of brain tissue sections reveals the binding of ANS to fibrillar deposits of amyloid peptide (Aβ) in amyloid plaques and in cerebrovascular amyloid. ANS imaging also highlights non-amyloid deposits of glial fibrillary acidic protein in brain tumors. Cultured cells under normal growth conditions possess a number of ANS-binding structures. High levels of ANS fluorescence are associated with the endoplasmic reticulum (ER), Golgi, and lysosomes-regions of protein folding and degradation. Nuclei are virtually devoid of ANS binding sites. Additional ANS binding is triggered by hyperthermia, thermal lesioning, proteasome inhibition, and induction of ER stress. We also use multiphoton imaging of ANS binding to follow the in vivo recovery of cells from protein-damaging insults over time. We find that ANS fluorescence tracks with the binding of the molecular chaperone Hsp70 in compartments where Hsp70 is present. ANS highlights the sensitivity of specific cellular targets, including the nucleus and particularly the nucleolus, to thermal stress and proteasome inhibition. Multiphoton imaging of ANS binding should be a useful probe for monitoring protein misfolding stress in cells.
Article: Laser and intense pulsed light therapy for the esthetic treatment of lower extremity veins.[show abstract] [hide abstract]
ABSTRACT: The role of lasers and intense pulsed light sources has gained increasing popularity over the last decade. Major advances associated with improved results are the main reasons associated with this increasing popularity. These advances include epidermal cooling technologies, variable spot sizes and pulse durations as well as the ability to deliver high-energy fluences. These advances have allowed the delivery of sufficient energy to cause uniform pan-endothelial necrosis without affecting epidermal structures causing adverse sequelae such as post-inflammatory hyperpigmentation and epidermal surface irregularities. The advent of extended-pulse longer-wavelength technologies such as the 1064 Neodymium : Yttrium Aluminum Garnet (Nd : YAG) laser have allowed the treatment of individuals with darker phenotypic skin types as well as deep blue reticular veins up to 3mm in diameter in a monomodal fashion. Combined approaches of sclerotherapy plus laser treatments performed at the same treatment session may produce synergistic results in selected individuals.American Journal of Clinical Dermatology 02/2003; 4(8):545-54. · 1.71 Impact Factor
Cytogenetics 02/1967; 6(1):1-19.
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ABSTRACT: The REF family of evolutionarily conserved heterogeneous ribonucleoprotein (hnRNP)-like proteins consists of one central RNP-type RNA binding domain flanked by Arg-Gly-rich regions of variable length. Members of this protein family bind directly to RNA and the mRNA export factor TAP/Mex67p, and it has been suggested that they facilitate the recruitment of TAP/Mex67p to cellular mRNPs. We show that the variable regions are necessary for binding of REFs to RNA and to TAP. Antibodies specific to REFs prevent their interaction with RNA in vitro. After microinjection into Xenopus oocytes, these antibodies inhibit mRNA nuclear export. This inhibition of export is observed whether or not the mRNAs are generated by splicing. The antibodies do not interfere with pre-mRNA splicing or with the nuclear export of constitutive transport element (CTE)-containing RNAs (directly mediated by TAP), so REF proteins must play a critical role in mRNA nuclear export, acting downstream of splicing and upstream of TAP/Mex67p. We also show that recombinant REFs stimulate directly the export of mRNAs that are otherwise exported inefficiently. Together, our data indicate that REFs are directly implicated in the export of mRNAs from the nucleus. More generally, we show that spliced and unspliced mRNAs use common export factors to reach the cytoplasm.Proceedings of the National Academy of Sciences 02/2001; 98(3):1030-5. · 9.68 Impact Factor