Suppression of src-induced transformed phenotype by expression of tropomyosin-1

Suppression of high M(r) tropomyosins (TMs) is a common feature of transformed cells. Previous work from this laboratory has demonstrated that the isoform 1 of TM, TM1, acts as an anti-oncogene in ras-transformed murine fibroblasts. In this study, we have investigated whether TM1 is a ras-specific suppressor, or a general suppressor protein of the cellular transformation. V-src transformed fibroblasts, which express decreased TM1, were transduced with a full-length cDNA to overexpress TM1. Both the control and the transduced cells expressed v-src kinase at comparable levels. TM1 expressing (src-T1) cells grew at a lower rate in monolayer, exhibited well spread, flat morphology than the control cells. Enhanced expression of TM1 resulted in improved microfilamental architecture. More significantly, src-T1 cells completely failed to grow under anchorage independent conditions. These data demonstrate that TM1 is as an anti-oncogene of functionally diverse oncogenes, and it is a class II tumor suppressor protein.     

Prevalence of occupational asthma in silk filatures

A clinical survey in two silk filatures revealed that 36.2% of the persons engaged in the processing of natural silk were suffering from bronchial asthma, while 16.9% of the total subjects had asthma of occupational origin. Skin prick tests using crude silkworm cocoon and pupal allergen extracts revealed that 28.8% of the subjects were sensitive to the silkworm-derived allergens. IgE antibodies specific to both cocoon and pupal allergens were demonstrable by RAST in the sera of patients with positive skin reactions and occupational asthma.     

Attenuation of cytomegalovirus-induced endothelial intercellular adhesion molecule-1 mRNA/protein expression and T lymphocyte adhesion by a 2'-O-methoxyethyl antisense oligonucleotide

BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) is strongly induced under inflammatory conditions associated with allograft rejection, thereby promoting leukocyte recruitment and activation at the site of inflammation. Enhancement of ICAM-1 expression can also be the result of viral infection, in particular human cytomegalovirus (CMV), a frequent source of complications in the transplant recipient. In vitro studies have shown that CMV infection of endothelial cells (EC) results in the direct enhancement of ICAM-1 expression and consequent leukocyte adhesion/activation suggesting mechanisms by which CMV exacerbates graft vascular disease. Although treatment of EC with ICAM-1-specific antisense oligonucleotides has been shown to attenuate ICAM-1 induction under simulated inflammatory conditions (i.e., TNF-alpha), no studies have addressed their effectiveness on virally-induced ICAM-1 expression. RESULTS: In the current investigation, we show that the progressive increase in endothelial ICAM-1 protein expression that follows inoculation with CMV correlates with a progressive accumulation of ICAM-1 mRNA. Furthermore, we demonstrate that treatment of EC with a partially 2'-O-methoxyethyl modified ICAM-1-specific antisense oligonucleotide before viral inoculation significantly reduces CMV-associated induction of ICAM-1 protein and mRNA expression. Finally, we show that antisense-mediated attenuation in ICAM-1 expression results in a significant reduction of T lymphocyte adhesion to CMV-infected EC monolayers, an interaction that has been implicated in allogeneic T lymphocyte activation, in viral transmission to transiently adherent leukocytes and subsequent hematogenous dissemination. CONCLUSIONS: These findings demonstrate for the first time that antisense oligonucleotides can effectively reverse virally-induced host cellular protein expression, specifically ICAM-1, as well as consequent T lymphocytes adhesion, thus broadening the potential clinical utility of antisense oligonucleotides.     

Toxicity of the sesquiterpene lactone parthenin to cultured bovine kidney cells.

The cytotoxic effect of the sesquiterpene lactone, parthenin was studied IN VITRO using cultured bovine kidney cells. The drug inhibited macromolecular synthesis. Fifty percent inhibitions in RNA, DNA and protein synthesis were observed when the cells were treated with parthenin at a final concentration of 1 microg/ml. The cytotoxic effect of parthenin was also evident by its ability to markedly inhibit the activities of key cellular enzymes after treatment of the cells with the toxin for 24 h.     

Generation of human monoclonal antibody-producing cell lines by Epstein-Barr virus (EBV)-transformation of B lymphocytes and somatic cell hybridization techniques

Summary The use of Epstein-Barr virus to immortalize human B cells and generate monoclonal antibody-producting B cell lines is described.     

Complete sequence of the genes encoding the VH and VL regions of low- and high-affinity monoclonal IgM and IgA1 rheumatoid factors produced by CD5+ B cells from a rheumatoid arthritis patient.

We have characterized the VH and VL genes of three low-affinity polyreactive and two high-affinity monoreactive IgM and IgA1 rheumatoid factor (RF) mAb generated using circulating CD5+ B cells from a single rheumatoid arthritis patient. We found that four and one RF mAb utilized genes of the VHIV and VHIII families, respectively. The VHIV gene usage by these RF mAb differs from the preferential VHIII, VHI, and, to a lesser extent, VHII gene usage by the IgM with RF activity found in patients with mixed cryoglobulinemia, Waldenstrom's macroglobulinemia, and other monoclonal gammopathies. In addition, in contrast to the preponderant kappa L chain usage by the RF in these patients, a lambda L chain was utilized by all RF mAb from our rheumatoid arthritis patient. Two RF mAbs utilized V lambda I, two V lambda IV, and one V lambda III L chains. The VH genes of the two low-affinity polyreactive IgM RF mAb were in germline configuration. When compared with the deduced amino acid sequence of the putatively corresponding genomic segment, the VH gene of the high-affinity monoreactive IgM RF mAb displayed five amino acid differences, all of which are in the complementarity determining regions (CDR), possibly the result of a process of somatic point mutation and clonal selection driven by Ag. The unavailability of the corresponding genomic VH segment sequences made it impossible to infer whether the VH genes utilized by the two IgA1 RF were in a germline or somatically mutated configuration. Sequencing of the genes encoding the H chain CDR3 (D segments) revealed that all three low-affinity polyreactive RF mAb displayed a much longer D segment (36-45 bases) than their high-affinity monoreactive counterparts (15-24 bases), raising the possibility that a long D segment may be one of the factors involved in antibody polyreactivity.     

Tau stabilizes microtubules by binding at the interface between tubulin heterodimers

The structure, dynamic behavior, and spatial organization of microtubules are regulated by microtubule-associated proteins. An important microtubule-associated protein is the protein Tau, because its microtubule interaction is impaired in the course of Alzheimer’s disease and several other neurodegenerative diseases. Here, we show that Tau binds to microtubules by using small groups of evolutionary conserved residues. The binding sites are formed by residues that are essential for the pathological aggregation of Tau, suggesting competition between physiological interaction and pathogenic misfolding. Tau residues in between the microtubule-binding sites remain flexible when Tau is bound to microtubules in agreement with a highly dynamic nature of the Tau–microtubule interaction. By binding at the interface between tubulin heterodimers, Tau uses a conserved mechanism of microtubule polymerization and, thus, regulation of axonal stability and cell morphology.Microtubules regulate cell division, cell morphology, intracellular transport, and axonal stability and, therefore, play crucial roles in cell function (1). Microtubules are built from tubulin heterodimers that polymerize into protofilaments and associate laterally into microtubules (2). Microtubule dynamics in neurons is modulated by several accessory proteins termed microtubule-associated proteins (3). However, little is known about the mechanism of assembly and stabilization of microtubules by microtubule-associated proteins.An important microtubule-associated protein is the protein Tau, which promotes formation of axonal microtubules, stabilizes them, and drives neurite outgrowth (4, 5). The adult human brain contains six isoforms of Tau, which are generated from a single gene by alternative splicing. The six isoforms are composed of either three or four repeats, with up to two N-terminal inserts, and range from 37 to 45 kDa (6). The 31- to 32-residue-long imperfect repeats are located in the carboxyl-terminal half of Tau and are highly conserved in several microtubule-associated proteins (7). The repeat domain is flanked by a proline-rich region that enhances binding to microtubules and microtubule assembly (8). Tau isoforms are developmentally regulated and have similar levels in the adult human brain (9).Impaired interaction of Tau with microtubules plays an important role in the pathology of several neurodegenerative diseases (10, 11). Dysregulation by genetic mutation or hyperphosphorylation affects the Tau–microtubule complex, leads to Tau detachment, causes instability and disassembly of microtubules, and, thus, perturbs axonal transport (12, 13). Microtubule-stabilizing drugs might therefore improve neuronal degeneration (14). When detached from microtubules, Tau can self-aggregate into insoluble aggregates through its hexapeptide motifs in the repeat domain (15). The deposition of aggregated Tau into neurofibrillary tangles and neuritic Tau pathology is one of the hallmarks in Alzheimer’s disease (16).Biochemical studies have shown that the repeat domain and the neighboring basic proline-rich region contribute strongly to microtubule binding (8, 17). In addition, a variety of binding sites and models of the Tau–microtubule complex were proposed (18–22). The models include binding of Tau to the outer surface of microtubules connecting tubulin subunits either across or along protofilaments (18). Tau might also reach into the interior of the microtubule wall near the binding site of the anticancer drug paclitaxel (19). Often these studies are complicated by the flexibility of the Tau protein, which belongs to the class of intrinsically disordered proteins (23, 24).Here, we studied the molecular mechanism of the interaction of Tau with microtubules by using a combination of NMR spectroscopy and mass spectrometry. We show that small groups of evolutionary conserved Tau residues bind dynamically at the interface between tubulin heterodimers, thereby promoting microtubule assembly and stabilization.