Typing the histogenetic origin of the tumor cells of lymphocyte-rich classical Hodgkin's lymphoma in relation to tumor cells of classical and lymphocyte-predominance Hodgkin's lymphoma.
(73/435)
Hodgkin's lymphoma (HL) is separated into the classical (c) and lymphocyte-predominance (lp) forms. Whereas classical Hodgkin-Reed/Sternberg (HRS) cells carry mutated immunoglobulin (Ig) gene rearrangements that are often "crippled" and lack intraclonal diversity, and are likely derived from preapoptotic germinal center (GC) B cells, the lymphocytic and histiocytic cells of lpHL are presumably derived from selected GC B cells and often show ongoing somatic hypermutation. The recently identified lymphocyte-rich classical (lrc) HL is characterized by HRS cells with the immunophenotype of classical HRS cells (CD30(+)CD15(+)CD20(-)CD45(-)) but an infiltrate similar to lpHL and a clinical behavior resembling lpHL. To identify the histogenetic origin of the HRS cells in lrcHL and to determine the relationship to the lymphoma cells of cHL and lpHL we characterized seven cases of lrcHL by immunohistochemistry and sequenced the rearranged Ig genes of single micromanipulated HRS cells. The expression patterns of BCL6, CD138, Oct2, and BOB1 in HRS cells of lrcHL showed differences to those of both cHL and lpHL. Analyses of rearranged Ig genes identified clonal HRS cell expansions carrying mutated Ig rearrangements without significant intraclonal diversity in all seven of the cases. In two cases crippling mutations, rendering originally functional V gene rearrangements nonfunctional, were observed. Thus, the mutation pattern of rearranged Ig genes of HRS cells in lrcHL is clearly different from those in lymphocytic and histiocytic cells of lpHL, and resembles the pattern in HRS cells of cHL, suggesting that HRS cells in lrcHL derive from (preapoptotic) GC B cells that silenced hypermutation. In one case in addition to the dominant HRS cell clone, CD30(+) EBV-infected HRS-like cells unrelated to the tumor clone were observed, suggesting development of an expanded population of EBV-harboring HRS-like cells in the microenvironment of HL. (+info)
Mechanical elongation of the centromere in the barley metaphase chromosome.
(74/435)
The present study investigated the mechanical elongation of the centromere in the barley chromosomes by a microneedle manipulation method for the structural analysis of the chromosomes. Chromosomes were extracted from barley root cells, affixed on a cover slip by a standard preparation method, and elongated in either distilled water, phosphate buffered saline (PBS), or 2 x sodium saline citrate (SSC). The mechanical property of the chromosome elongation was assessed by the measurement of the force required for the elongation of chromosomes. This assessment has shown that the chromosomes in distilled water were much firmer than those in the PBS or 2 x SSC. To confirm the elongation of the centromere, the elongated chromosomes were investigated by fluorescence in situ hybridization with a centromere probe. The fluorescence information indicated that the extent of the loosening of the centromere during elongation differed depending on the buffers used; the centromere elongated in 2 x SSC was more loosened than that in the PBS. Atomic force microscopy also revealed the structure of the unpacked centromere after the mechanical elongation, when rows of fibrous structures about 30 to 50 nm thick were clearly observed in the centromere elongated in 2 x SSC. The investigation of elongated chromosomes should prove useful for an understanding of the structural analysis of chromosomes. (+info)
Atomic force microscope-based dissection of human metaphase chromosomes and high resolutional imaging by carbon nanotube tip.
(75/435)
The present study was performed to introduce a novel chromosome dissection method employing atomic force microscopy (AFM) in a dynamic force mode for the chemical or molecular biological analysis of tiny chromosomal fragments. After AFM observation of human chromosomes prepared for light microscopy, a region of interest was dissected by increasing the loading force in a series of single-line scans of the target portion by controlling it with the amplitude reference of the tip in a dynamic force mode. The marker gene of the nucleolar organizing region (NOR) was amplified by our designed primers for 5.8S ribosomal DNA. After the dissection, topographic profiles in the section were then obtained with a carbon nanotube (CNT) probe in ambient condition. These results are discussed in relation to a fundamental technology for chromosomal analysis. (+info)
Combined optical trapping and single-molecule fluorescence.
(76/435)
BACKGROUND: Two of the mainstay techniques in single-molecule research are optical trapping and single-molecule fluorescence. Previous attempts to combine these techniques in a single experiment - and on a single macromolecule of interest - have met with little success, because the light intensity within an optical trap is more than ten orders of magnitude greater than the light emitted by a single fluorophore. Instead, the two techniques have been employed sequentially, or spatially separated by distances of several micrometers within the sample, imposing experimental restrictions that limit the utility of the combined method. Here, we report the development of an instrument capable of true, simultaneous, spatially coincident optical trapping and single-molecule fluorescence. RESULTS: We demonstrate the capability of the apparatus by studying force-induced strand separation of a rhodamine-labeled, 15 base-pair segment of double-stranded DNA, with force applied perpendicular to the axis of the DNA molecule. As expected, we observed abrupt mechanical transitions corresponding to the unzipping of DNA at a critical force. Transitions occurred concomitant with changes in the fluorescence of dyes attached at the duplex ends, which became unquenched upon strand separation. CONCLUSIONS: Through careful optical design, the use of high-performance spectral notch filters, a judicious choice of fluorophores, and the rapid acquisition of data gained by computer-automating the experiment, it is possible to perform combined optical trapping and single-molecule fluorescence. This opens the door to many types of experiment that employ optical traps to supply controlled external loads while fluorescent molecules report concurrent information about macromolecular structure. (+info)
Kinetics from nonequilibrium single-molecule pulling experiments.
(77/435)
Mechanical forces exerted by laser tweezers or atomic force microscopes can be used to drive rare transitions in single molecules, such as unfolding of a protein or dissociation of a ligand. The phenomenological description of pulling experiments based on Bell's expression for the force-induced rupture rate is found to be inadequate when tested against computer simulations of a simple microscopic model of the dynamics. We introduce a new approach of comparable complexity to extract more accurate kinetic information about the molecular events from pulling experiments. Our procedure is based on the analysis of a simple stochastic model of pulling with a harmonic spring and encompasses the phenomenological approach, reducing to it in the appropriate limit. Our approach is tested against computer simulations of a multimodule titin model with anharmonic linkers and then an illustrative application is made to the forced unfolding of I27 subunits of the protein titin. Our procedure to extract kinetic information from pulling experiments is simple to implement and should prove useful in the analysis of experiments on a variety of systems. (+info)
Tensile force-dependent neurite elicitation via anti-beta1 integrin antibody-coated magnetic beads.
(78/435)
Previous work using glass microneedles to apply calibrated, localized force to neurons showed that tensile force is a sufficient signal for neurite initiation and elongation. However, previous studies did not examine the kinetics or probability of neurite initiation as a function of force or the rate of force application. Here we report the use of a new technique-magnetic bead force application-to systematically investigate the role of force in these phenomena with better ease of use and control over force than glass microneedles. Force-induced neurite initiation from embryonic chick forebrain neurons appeared to be a first-order random process whose rate increased with increasing force, and required the presence of peripheral microtubules. In addition, the probability of initiation was more than twofold lower for neurons exposed to rapid initial force ramps (450 pN/s) than for neurons exposed to slower ramps (1.5 and 11 pN/s). We observed a low force threshold for elongation (15-100 pN), which was not previously detected in chick forebrain neurites elongated by glass microneedles. Finally, neurites subjected to constant force elongated at variable instantaneous rates, and switched abruptly between elongation and retraction, similar to spontaneous, growth-cone-mediated outgrowth and microtubule dynamic instability. (+info)
Shrinking the biologic world--nanobiotechnologies for toxicology.
(79/435)
Although toxicologic effects need to be considered at the organismal level, the adverse events originate from interactions and alterations at the molecular level. Cellular structures and functions can be disrupted by modifications of the nanometer structure of critical molecules; therefore, devices used to assess biologic and toxicologic processes at the nanoscale will allow important new research pursuits. In order to properly assess alterations at these dimensions, nanofabricated tools are needed to detect, separate, analyze, and manipulate cells or biologic molecules of interest. The emergence of laser tweezers, surface plasmon resonance (SPR), laser capture microdissection (LCM), atomic force microscopy (AFM), and multi-photon microscopes have allowed for these assessments. Micro- and nanobiotechnologies will further advance biologic, clinical, and toxicologic endeavors with the aid of miniaturized, more sensitive devices. Miniaturized table-top laboratory equipment incorporating additional innovative technologies can lead to new advances, including micro total analysis systems (microTAS) or "lab-on-a-chip" and "sentinel sensor" devices. This review will highlight several devices, which have been made possible by techniques originating in the microelectronics industry. These devices can be used for toxicologic assessment of cellular structures and functions, such as cellular adhesion, signal transduction, motility, deformability, metabolism, and secretion. (+info)
Profiling of Hodgkin's lymphoma cell line L1236 and germinal center B cells: identification of Hodgkin's lymphoma-specific genes.
(80/435)
The malignant cells of classical Hodgkin's lymphoma (cHL), Hodgkin and Reed-Sternberg (HRS) cells, appear to be derived from germinal center (GC) B cells in most cases of the disease. Apart from recent findings of constitutive activation of some transcription factors and autocrine stimulation by cytokine receptors, the mechanisms of malignant transformation in cHL still remain poorly understood. We performed a large scale gene expression study using serial analysis of gene expression (SAGE), comparing the cHL cell line L1236 and human GC B cells. Semiquantitative RT-PCR was used to confirm results from the SAGE and to analyze gene expression in 3 additional cHL cell lines. To investigate expression of some genes in cHL cases, we applied RT-PCR on microdissected HRS cells. In total, 464 genes showed a change in expression level of 5-fold or higher. For 12 genes (out of 177) identified as upregulated in L1236 cells, RT-PCR confirmed the SAGE results and also showed elevated expression in 3 other cHL cell lines. For 3 of the upregulated genes, expression by HRS cells in the tissue also was confirmed. Several of the differentially expressed genes may play a role in the pathogenesis of cHL because they represent potential oncogenes, such as rhoC, L-myc, and PTP4A, or transcription factors, such as ATF-5, ATBF1, and p21SNFT. The genes that showed significantly deregulated expression in HRS cells should be helpful not only for the identification of genes involved in the pathogenesis of cHL but also for discovering potential prognostic markers or therapeutic targets. (+info)