Abstract Human promyelocytic cells (HL-60) were labeled with 35S-sulfate and either 3H-glucosamine or 3H-serine as precursors. Accumulation of 35S-labeled macromolecules was approximately linear for up to 96 h, with a mean cell:medium ratio of 5.5:1, although activity/105 viable cells reached a plateau level after 24 h. Virtually none of the cell-associated proteoglycan was removed by trypsinization, consistent with a predominantly intracellular localization. Proteoglycan heterogeneity was investigated by DEAE-Sephacel chromatography, isopyknic CsCI gradient centrifugation, and gel filtration chromatography. HL-60 cells appeared to synthesize a single proteoglycan species, Kav = 0.46 on Sepharose CL-4B and Kav = 0.32 on Sepharose CL-6B, recovered primarily from the high-density fractions of a dissociative CsCI gradient (ρ > 1.40 g/l). Degradation products of lower charge density, lower buoyant density, and lower hydrodynamic size were also present, mainly in the cell pellets. The major proteoglycan was found to contain chondroitin sulfate chains of average Mr = 14.5 kD, yielding virtually 100% 4-sulfated disaccharides on digestion with chondroitinase ABC. The proteoglycan was resistant to trypsin, chymotrypsin, plasmin, and papain, and the core protein Mr was approximately 20 kD by molecular sieve chromatography. Induction of HL-60 cells with 0.15 dimethyl sulfoxide (DMSO) resulted in differentiation to a more mature granulocytic phenotype and was associated with a reduction in 35S-sulfate incorporation to 45% of control values or 32%, expressed as activity/105 cells. Proteoglycans synthesized by DMSO-treated cells were identical to those from untreated cells in terms of hydrodynamic size, glycosaminoglycan Mr, and sulfation.
Correct human β-globin mRNA has been restored in erythroid cells from transgenic mice carrying the human gene with β-globin IVS2–654 splice mutation and from thalassemia patients with the IVS2–654/βE genotype. This was accomplished in a dose- and time-dependent manner by free uptake of morpholino oligonucleotide antisense to the aberrant splice site at position 652 of intron 2 in β-globin pre-mRNA. Under optimal conditions of oligonucleotide uptake, the maximal levels of correct human β-globin mRNA and hemoglobin A in patients9 erythroid cells were 77 and 54%, respectively. These levels of correction were equal to, if not higher than, those obtained by syringe loading of the oligonucleotide into the cells. Comparison of splicing correction results with the cellular uptake of fluorescein-labeled oligonucleotide indicated that the levels of mRNA and hemoglobin A correlate well with the nuclear localization of the oligonucleotide and the degree of erythroid differentiation of cultured cells. Similar but not as pronounced results were obtained after the oligonucleotide treatment of bone marrow cells from IVS2–654 mouse. The effectiveness of the free antisense morpholino oligonucleotide in restoration of correct splicing of IVS2–654 pre-mRNA in cultured erythropoietic cells from transgenic mice and thalassemic patients suggests the applicability of this or similar compounds in in vivo experiments and possibly in treatment of thalassemia.
Abstract Previous work has shown that dendritic cells (DCs) express specific chemokine receptors that allow for coordinated movement in vivo. To test the in vivo relevance of this, we used a murine melanoma system and knockout mice to investigate the function of the chemokine receptor CCR5 and its ligands, CCR ligand (CCL)3 and CCL5. We found that the lack of CCR5 in the host mouse resulted in delayed tumor growth, but this effect was overcome at a higher tumor load. With the administration of tumor charged DCs, CCR5−/− mice that had previously been injected with tumor were completely protected from tumor. This effect was dependent on the dose of tumor cells and the expression of CCR5 on the DC and its absence in the host. In contrast, the loss of the CCR5 ligand, CCL3, led to an early delay in tumor growth that did not persist, while the absence of the CCR5 ligand, CCL5, had no effect. Blocking the activity of CCR5 in the host may represent a new strategy for enhancing the activity of a therapeutic melanoma DC vaccine.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTOptimization of separation number in gas chromatography with fused silica capillary columns under temperature programmed conditionsLouis A. Jones, Suzanne L. Kirby, Cheryl L. Garganta, Thomas M. Gerig, and James D. MulikCite this: Anal. Chem. 1983, 55, 8, 1354–1360Publication Date (Print):July 1, 1983Publication History Published online1 May 2002Published inissue 1 July 1983https://pubs.acs.org/doi/10.1021/ac00259a038https://doi.org/10.1021/ac00259a038research-articleACS PublicationsRequest reuse permissionsArticle Views112Altmetric-Citations14LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Despite a lack of conclusive evidence, many researchers in the field view computer-assisted instruction (CAI) as an opportunity for improved instruction for students with disabilities. This study examined the effects of a CAI program, Lexia Strategies for Older Students (SOS)™ on the word recognition skills of four, upper elementary students with mild disabilities. This study used a multiple-probe design across three targeted reading skill conditions per student. Findings revealed that some students were able to meet mastery of basic word reading skills with Lexia SOS alone, while others needed additional direct instruction. Student perceptions of Lexia SOS were positive. Results have particular implications for instruction in classrooms beyond the primary grades (K-3) when the focus of the curriculum shifts away from basic decoding instruction. Directions for future research are discussed.
Abstract The Steel anemia of mice results from an inherited defect in the hematopoietic microenvironment. Proteoglycans synthesized by bone marrow stromal cells are an important functional component of the hematopoietic microenvironment in normal animals. It is thus possible that Steel anemia results from a molecular abnormality involving bone marrow stromal proteoglycans. To investigate this possibility, we studied proteoglycan synthesis in three stromal cell lines from Steel anemic (SI/SI d ) animals and two control stromal cell lines, one (+/+2.4) from a non‐anemic littermate, and one (GBI/6) from a normal mouse. Proteoglycans were precursor labelled with 35 S sulfate and separated by ion exchange HPLC, CsCI density gradient centrifugation, and molecular sieve HPLC. Glycosaminoglycan (GAG) moieties were characterized by molecular sieve HPLC and enzyme sensitivity. There were no consistent differences in total proteoglycan synthesis, proteoglycan heterogeneity, GAG hydrodynamic size, or enzyme sensitivity among the cell lines studied. Growth factor binding to stromal extracellular matrix (ECM) was studied by co‐culture of an IL‐3‐dependent cell line (FDC‐P1) with cell‐free ECM preparations from an SI/SI d and a control (GBI/6) stromal cell line, with and without pre‐incubation with IL‐3. Cell‐free ECM preparations from SI/SI d and control cell lines supported FDC‐P1 growth to an approximately equal extent after pre‐incubation with II‐3. FDC‐P1 growth support by ECM preparations from both cell lines was also observed without IL‐3 pre‐incubation, although to a lesser extent, suggesting ECM binding of endogenous growth factors synthesized by the stromal cells.
In multiple myeloma, karyotypic 14q32 translocations have been identified at a variable frequency (10–60% in different studies). In the majority of cases, the partner chromosome has not been identified (14q+), and in the remaining cases, a diverse array of chromosomal partners has been implicated, with 11q13 being the most common. We developed a comprehensive Southern blot assay to identify and distinguish different kinds of immunoglobulin heavy chain (IgH) switch recombination events. Illegitimate switch recombination fragments (defined as containing sequences from only one switch region) are potential markers of translocation events into IgH switch regions and were identified in 15 of 21 myeloma cell lines, including seven of eight karyotyped lines that have no detectable 14q32 translocation. From all nine lines or tumor samples analyzed further, cloned illegitimate switch recombination fragments were confirmed to be IgH switch translocation breakpoints. In three of these cases, the translocation breakpoint was shown to be present in the primary tumor. These translocation breakpoints involve six chromosomal loci: 4p16.3 (two lines and the one tumor); 6; 8q24.13; 11q13.3 (in three lines); 16q23.1; and 21q22.1. We suggest that translocations into the IgH locus ( i ) are frequent (karyotypic 14q32 translocations and/or illegitimate switch recombination fragments are present in primary tumor samples and in 19 of 21 lines that we have analyzed); ( ii ) occur mainly in switch regions; and ( iii ) involve a diverse but nonrandom array (i.e., frequently 11q13 or 4p16) of chromosomal partners. This appears to be the most frequent genetic abnormality in multiple myeloma.
