Direct visualization of antigen-specific T cells: HTLV-1 Tax11–19- specific CD8+ T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients

Human T lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11–19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2–Ig complex to directly visualize HTLV-1 Tax11–19-specific T cells from peripheral blood and cerebrospinal fluid without in vitro stimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8+ lymphocytes specific for the HTLV-1 Tax11–19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11–19-specific T cells in the cerebrospinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11–19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11–19-specific CD8+ T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-α and γ-interferon depending on the severity of the disease. Thus, visualization of antigen-specific T cells demonstrates that HTLV-1 Tax11–19-specific CD8+ T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.

Direct Involvement of N-Cadherin–mediated Signaling in Muscle Differentiation

Cell–cell interactions, mediated by members of the cadherin family of Ca2+-dependent adhesion molecules, play key roles in morphogenetic processes as well as in the transduction of long-range growth and differentiation signals. In muscle differentiation cell adhesion is involved in both early stages of myogenic induction and in later stages of myoblast interaction and fusion. In this study we have explored the involvement of a specific cadherin, namely N-cadherin, in myogenic differentiation. For that purpose we have treated different established lines of cultured myoblasts with beads coated with N-cadherin–specific ligands, including a recombinant N-cadherin extracellular domain, and anti-N-cadherin antibodies. Immunofluorescent labeling for cadherins and catenins indicated that treatment with the cadherin-reactive beads for several hours enhances the assembly of cell–cell adherens-type junctions. Moreover, immunofluorescence and immunoblotting analyses indicated that treatment with the beads for 12–24 h induces myogenin expression and growth arrest, which are largely independent of cell plating density. Upon longer incubation with the beads (2–3 d) a major facilitation in the expression of several muscle-specific sarcomeric proteins and in cell fusion into myotubes was observed. These results suggest that surface clustering or immobilization of N-cadherin can directly trigger signaling events, which promote the activation of a myogenic differentiation program.

Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary carcinoma, a unique animal model for human bronchioalveolar carcinoma. We previously isolated a JSRV proviral clone and showed that it was both infectious and oncogenic. Thus JSRV is necessary and sufficient for the development of ovine pulmonary carcinoma, but no data are available on the mechanisms of transformation. Inspection of the JSRV genome reveals standard retroviral genes, but no evidence for a viral oncogene. However, an alternate ORF in pol (orf-x) might be a candidate for a transforming gene. We tested whether the JSRV genome might encode a transforming gene by transfecting an expression plasmid for JSRV [pCMVJS21, driven by the cytomegalovirus (CMV) immediate early promoter] into mouse NIH 3T3 cells. Foci of transformed cells appeared in the transfected cultures 2–3 weeks posttransfection; cloned transformants showed anchorage independence for growth, and they expressed JSRV RNA. These results indicate that the JRSV genome contains information with direct transforming potential for NIH 3T3 cells. Transfection of a mutated version of pCMVJS21 in which the orf-x protein was terminated by two stop codons also gave transformed foci. Thus, orf-x was eliminated as the candidate transforming gene. In addition, another derivative of pCMVJS21 (pCMVJS21ΔGP) in which the gag, pol (and orf-x) coding sequences were deleted also gave transformed foci. These results indicate that the envelope gene carries the transforming potential. This is an unusual example of a native retroviral structural protein with transformation potential.

Direct conversion of an oligopeptide from a β-sheet to an α-helix: A model for amyloid formation

A 16-amino acid oligopeptide forms a stable β-sheet structure in water. In physiological solutions it is able to self-assemble to form a macroscopic matrix that stains with Congo red. On raising the temperature of the aqueous solution above 70°C, an abrupt structural transition occurs in the CD spectra from a β-sheet to a stable α-helix without a detectable random-coil intermediate. With cooling, it retained the α-helical form and took several weeks at room temperature to partially return to the β-sheet form. Slow formation of the stable β-sheet structure thus shows kinetic irreversibility. Such a formation of very stable β-sheet structures is found in the amyloid of a number of neurological diseases. This oligopeptide could be a model system for studying the protein conformational changes that occurs in scrapie or Alzheimer disease. The abrupt and direct conversion from a β-sheet to an α-helix may also be found in other processes, such as protein folding and protein–protein interaction. Furthermore, such drastic structure changes may also be exploited in biomaterials designed as sensors to detect environmental changes.

Direct Measurement of Calcium Transport across Chloroplast Inner-Envelope Vesicles1

The initial rate of Ca2+ movement across the inner-envelope membrane of pea (Pisum sativum L.) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Ca2+-sensitive fluorophore fura-2. Calibration of fura-2 fluorescence was achieved by combining a ratiometric method with Ca2+-selective minielectrodes to determine pCa values. The initial rate of Ca2+ influx in predominantly right-side-out inner-envelope membrane vesicles was greater than that in largely inside-out vesicles. Ca2+ movement was stimulated by an inwardly directed electrochemical proton gradient across the membrane vesicles, an effect that was diminished by the addition of valinomycin in the presence of K+. In addition, Ca2+ was shown to move across the membrane vesicles in the presence of a K+ diffusion potential gradient. The potential-stimulated rate of Ca2+ transport was slightly inhibited by diltiazem and greatly inhibited by ruthenium red. Other pharmacological agents such as LaCl3, verapamil, and nifedipine had little or no effect. These results indicate that Ca2+ transport across the chloroplast inner envelope can occur by a potential-stimulated uniport mechanism.