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PubMed1994

The octameric histone core of the nucleosome. Structural issues resolved.

The crystal structure of the histone octamer has now been determined at 3.1 A resolution and refined to a crystallographic R value of 25.5%. The overall shape of the structure is significantly different from that originally reported by Burlingame et al. and its length is now in agreement with that observed by Klug et al. in their low-resolution studies. The experimental intensity data used in constructing the new electron density map were the same as those used by Burlingame et al. for the original electron density map. In addition, the methods used in producing the new density map were also the same as those for the original map. The only difference between the two calculations was the selection of the heavy-atom location. The large change seen in the structural image (110 A x 70 A x 70 A versus 55 A x 70 A x 70 A) was due to a relatively small change (2.27 A shift) of the heavy-atom site. The fact that the shape and size of the original structure were incorrect is surprising and unusual, since the electron density map that produced the original model was clear for most parts of the structure; one could easily see the well-formed right-handed helices of the H2A and H2B molecules, and the ordered parts of the H2A and H2B molecules could be easily traced from end to end. A comparison of the two maps shows that the original image was derived from two fused copies of the correct structure rotated by +/- 120 degrees from its true location along a rotation axis parallel to the z-axis and the image seen was a partial (about 19.5%) overlap of two molecules. An explanation is given as to how such a small shift of the heavy-atom position could create such a double image in the unit cell, and how the original electron density map could be converted to the new map by a phase modification in the Fourier synthesis. This study resolves the differences between the analyses of the shape and size of the histone octamer structure.

Crystallography, X-Ray↗
PubMed2003

Expression and distribution of distinct variants of E-MAP-115 during proliferation and differentiation of human intestinal epithelial cells.

Epithelial cell proliferation and differentiation occur concomitant with striking remodeling of the cytoskeleton. Microtubules (MTs) play important roles in these processes, during which the MTs themselves are reorganized and stabilized by microtubule-associated proteins (MAPs). Among the proteins classified as structural MAPs, E-MAP-115 (also named ensconsin) is preferentially expressed in cells of epithelial origin. The aims of this study were, first, to determine if E-MAP-115, like other MAPs, is expressed as different isoforms during differentiation and, second, to perform a detailed analysis of the expression and distribution of any E-MAP-115 variants detected in intestinal epithelial cells during their polarization/differentiation. It was our expectation that these data would help us to develop hypotheses concerning the role of this MAP in epithelial development. We report the expression of three E-MAP-115 transcripts encoding isoforms of 115, 105, and 95 kDa; two display an expression gradient inverse to the third one as Caco-2 cells progress from proliferation through the stages of differentiation. To monitor the proteins produced from each transcript, we used purified polyclonal antibodies against synthetic peptides contained within the 115, 105, and 95 kDa isoforms to assay proliferating and differentiating CaCo-2 cells. Our results indicate that the expression and MT-binding capacity of the 115, 105, and 95 kDa isoforms vary upon proliferation/differentiation of the cells. E-MAP-115 proteins colocalize with MTs in proliferative and differentiated Caco-2 cells; in vivo, they are expressed in both crypt and villus epithelial cells where they are mainly concentrated at the apical pole of the cells.

Humans↗
PubMed1992

Ewing's tumor X mouse hybrids expressing the MIC2 antigen: analyses using fluorescence CDD-banding and non-isotopic ISH.

We present a highly sensitive method that has been applied to map the chromosomal origin of the prominent cell surface antigen of Ewing's tumor cells recognized by monoclonal antibody HBA-71. The technique allows an unambiguous identification of human chromosomal material in interspecific cell hybrids. This is achieved by fluorescent in situ hybridization of biotinylated total human DNA, followed by high resolution fluorescence banding with the chromomycin/distamycin/DAPI triple stain. An advantage of this method is that all signals can be visualized in one single operation by simply switching the appropriate filter blocks. The protocol has proved extremely useful in gene mapping by means of interspecific cell hybrids, a technique that depends on the accurate and unambiguous recognition of the relevant (e.g., human) genetic material in the clonal genome. Our studies confirm that this antigen is the product of the MIC2 gene, which is so far the only well-studied pseudoautosomal gene in man, and which is located on the short arms of chromosomes X and Y. Furthermore, no influence of the Ewing's tumor-specific translocation t(11;22) on MIC2 expression could be discerned.

Animals↗
PubMed1997

Highly conservative reciprocal translocations formed by apparent joining of exchanged DNA double-strand break ends.

Chromosomal translocations induced by ionizing radiation and radiomimetic drugs are thought to arise by incorrect joining of DNA double-strand breaks. To dissect such misrepair events at a molecular level, large-scale, bleomycin-induced rearrangements in the aprt gene of Chinese hamster ovary D422 cells were mapped, the breakpoints were sequenced, and the original non-aprt parental sequences involved in each rearrangement were recovered from nonmutant cells. Of seven rearrangements characterized, six were reciprocal exchanges between aprt and unrelated sequences. Consistent with a mechanism involving joining of exchanged double-strand break ends, there was, in most cases, no homology between the two parental sequences, no overlap in sequences retained at the two newly formed junctions, and little or no loss of parental sequences (usually </=2 bp) at the breakpoints. The breakpoints were strongly correlated (P < 0.0001) with expected sites of bleomycin-induced, double-strand breaks. Fluorescence in situ hybridization indicated that, in six of the mutants, the rearrangement was accompanied by a chromosomal translocation at the aprt locus, because upstream and downstream flanking sequences were detected on separate chromosomes. The results suggest that repair of free radical-mediated, double-strand breaks in confluence-arrested cells is effected by a conservative, homology-independent, end-joining pathway that does not involve single-strand intermediate and that misjoining of exchanged ends by this pathway can directly result in chromosomal translocations.

Adenine Phosphoribosyltransferase↗
PubMed1994

Physical mapping of origins of replication in the fission yeast Schizosaccharomyces pombe.

We isolated four fragments from the Schizosaccharomyces pombe genome that mediate autonomous replication. A two-dimensional gel analysis revealed that in each case initiation could be mapped to within the S. pombe sequences. In three of the fragments, initiation could be mapped to one discrete location. In the fourth fragment, subcloning and two-dimensional gel analysis suggested that two discrete origins of replication were located within 3 kb of each other. When in proximity, usually only one of these origins fired, suggesting origin interference. Two-dimensional gel analysis of the four origin fragments at their genomic locations demonstrated that each is used in the chromosomes, but in only a subset of cells or cell divisions. The S. pombe genome appears to contain many discrete origins, not all of which fire in any given cell and some of which are closely spaced. Not I/Sfi I mapping of the five origins from this and a previous study indicates that they are randomly distributed throughout the genome and appear to be representative of chromosomal origins of replication in this organism. We compare the features of S. pombe replication origins with those of S. cerevisiae and animal cells.

Chromosome Mapping↗
PubMed2006

DNA replication origin interference increases the spacing between initiation events in human cells.

Mammalian DNA replication origins localize to sites that range from base pairs to tens of kilobases. A regular distribution of initiations in individual cell cycles suggests that only a limited number of these numerous potential start sites are converted into activated origins. Origin interference can silence redundant origins; however, it is currently unknown whether interference participates in spacing functional human initiation events. By using a novel hybridization strategy, genomic Morse code, on single combed DNA molecules from primary keratinocytes, we report the initiation sites present on 1.5 Mb of human chromosome 14q11.2. We confirm that initiation zones are widespread in human cells, map to intergenic regions, and contain sequence motifs found at other mammalian initiation zones. Origins used per cell cycle are less abundant than the potential sites of initiation, and their limited use increases the spacing between initiation events. Between-zone interference decreases in proportion to the distance from the active origin, whereas within-zone interference is 100% efficient. These results identify a hierarchical organization of origin activity in human cells. Functional origins govern the probability that nearby origins will fire in the context of multiple potential start sites of DNA replication, and this is mediated by origin interference.

Base Pairing↗
PubMed2000

The lymphatic endothelium of the avian wing is of somitic origin.

It has recently been shown that there are lymphangioblasts in the early avian wing bud, but fate map studies on the origin of these cells have not yet been performed. The lymphatics in the wings of 10-day-old chick and quail embryos are characterized by both the position along with all major blood vascular routes and by the Vascular Endothelial Growth Factor Receptor-3 (VEGFR-3) expression. In the quail, the endothelium of both blood vessels and lymphatics can be marked with the QH1 antibody. We have grafted the dorsal halves of epithelial somites of 2-day-old quail embryos homotopically into chick embryos. The grafting was performed at the wing level and the host embryos were reincubated until day 10. The chimeric wings were studied with the QH1 antibody alone and with double staining consisting of VEGFR-3 in situ hybridization and QH1 immunofluorescence. Our results show that in the wing the endothelium of both the blood vessels and the lymphatics is derived from the somites. QH1-positive endothelial cells form the vasculature of the chimeric wings. Chimeric lymphatics of the wing can be identified because of their typical position and their VEGFR-3 and QH1 double-positivity. This shows that not only the blood vascular cells but also the lymphatic endothelial cells of the avian wing are born in the paraxial/somitic mesoderm.

Animals↗
PubMed2003

Cardiac neural crest contributes to cardiomyogenesis in zebrafish.

In birds and mammals, cardiac neural crest is essential for heart development and contributes to conotruncal cushion formation and outflow tract septation. The zebrafish prototypical heart lacks outflow tract septation, raising the question of whether cardiac neural crest exists in zebrafish. Here, results from three distinct lineage-labeling approaches identify zebrafish cardiac neural crest cells and indicate that these cells have the ability to generate MF20-positive muscle cells in the myocardium of the major chambers during development. Fate-mapping demonstrates that cardiac neural crest cells originate both from neural tube regions analogous to those found in birds, as well as from a novel region rostral to the otic vesicle. In contrast to other vertebrates, cardiac neural crest invades the myocardium in all segments of the heart, including outflow tract, atrium, atrioventricular junction, and ventricle in zebrafish. Three distinct groups of premigratory neural crest along the rostrocaudal axis have different propensities to contribute to different segments in the heart and are correspondingly marked by unique combinations of gene expression patterns. Zebrafish will serve as a model for understanding interactions between cardiac neural crest and cardiovascular development.

Animals↗
PubMed2000

HLA-restricted immune response to mycobacterial antigens: relevance to vaccine design.

Identification of mycobacterial antigens that are recognized by CD4+ Th1 cells in HLA-nonrestricted manner or in association with multiple allelic products is required to develop universally effective vaccines against mycobacterial diseases. Our studies in this direction have shown that several recombinant mycobacterial antigens of cytosolic and culture filtrate origin are recognized by CD4+ Th1 cells. Mapping of T cell epitopes with overlapping synthetic peptides covering the entire sequence of these antigens identified peptide sequences stimulatory for Th1 cells. HLA-restriction analysis showed that in addition to HLA-DRB1 products (serologically defined HLA-DR1 to HLA-DR10), the HLA molecules encoded by HLA-DRB3 (HLA-DR52) and HLA-DRB4 (HLA-DR53) are important in presentation of mycobacterial antigens and epitopes to T cells. Depending on the T cell donor, the presentation of a given antigen or peptide could be restricted by HLA-DRB1, HLA-DRB3, and/or HLA-DRB4 products. In addition, stimulation of Th1 cells by some antigens and peptides in the presence of autologous and HLA-DR mismatched allogeneic APC suggested promiscuous presentation. These results taken together suggest that from HLA-restriction perspective, several mycobacterial antigens qualify as candidates for subunit or recombinant vaccine design against mycobacterial diseases.

Antigen Presentation↗
PubMed2005

Mesenchymal progenitor cells localize within hematopoietic sites throughout ontogeny.

Mesenchymal stem cells (MSCs) have great clinical potential for the replacement and regeneration of diseased or damaged tissue. They are especially important in the production of the hematopoietic microenvironment, which regulates the maintenance and differentiation of hematopoietic stem cells (HSCs). In the adult, MSCs and their differentiating progeny are found predominantly in the bone marrow (BM). However, it is as yet unknown in which embryonic tissues MSCs reside and whether there is a localized association of these cells within hematopoietic sites during development. To investigate the embryonic origins of these cells, we performed anatomical mapping and frequency analysis of mesenchymal progenitors at several stages of mouse ontogeny. We report here the presence of mesenchymal progenitors, with the potential to differentiate into cells of the osteogenic, adipogenic and chondrogenic lineages, in most of the sites harboring hematopoietic cells. They first appear in the aorta-gonad-mesonephros (AGM) region at the time of HSC emergence. However, at this developmental stage, their presence is independent of HSC activity. They increase numerically during development to a plateau level found in adult BM. Additionally, mesenchymal progenitors are found in the embryonic circulation. Taken together, these data show a co-localization of mesenchymal progenitor/stem cells to the major hematopoietic territories, suggesting that, as development proceeds, mesenchymal progenitors expand within these potent hematopoietic sites.

Adipocytes↗
PubMed1999

Familial pseudohyperkalemia maps to the same locus as dehydrated hereditary stomatocytosis (hereditary xerocytosis).

Familial pseudohyperkalemia is a "leaky red blood cell" condition in which the cells show a temperature-dependent loss of potassium (K) from red blood cells when stored at room temperature, manifesting as apparent hyperkalemia. The red blood cells show a reduced lifespan in vivo but there is no frank hemolysis. Studies of cation content and transport show a marginal increase in permeability at 37 degrees C and a degree of cellular dehydration, qualitatively similar to the changes seen in dehydrated hereditary stomatocytosis (hereditary xerocytosis). Physiological studies have shown that the passive leak to K has an abnormal temperature dependence, such that the leak is less sensitive to temperature than that in normal cells. We performed genetic mapping on the original family and found that the condition in this kindred maps to the same locus (16q23-ter) that we have previously identified for an Irish family with dehydrated hereditary stomatocytosis, which does not show the same temperature effects.

Anemia, Hemolytic, Congenital↗
PubMed1984

[Hepatoma 27 cells and the epithelium of the large intestine in rats contain the identical set of prekeratin proteins].

It has been shown by means of the immunoblot technique in combination with monoclonal antibodies and peptide mapping that hepatocytes, hepatoma 27 cells, and rat colon enterocytes exhibit a common prekeratin protein with a molecular weight of 49 kD (PK49). This protein and vimentin, a protein contained by intermediate filaments of mesenchymal cells, share at least one antigenic determinant. The generally accepted procedure for prekeratin purification leads to a more or less pronounced degradation of PK49. The degree of degradation is dependent on the type of the tissue extracted. High heterogenicity of prekeratin polypeptides described elsewhere might be due partly to such a degradation process. In addition to PK49, hepatoma 27 cells, absorbing, goblet and proliferating cells of the colon demonstrated three more prekeratin proteins: two major (PK55 and PK40) and one minor (PK53). Monoclonal antibodies not reacting with PK49 do not recognize PK55, PK53 and PK40. PK55, PK49 and PK40 of hepatoma 27 are identical to the appropriate proteins of the colonic epithelium as judged by peptide mapping. Thus, the cells of the hepatocyte origin are able to synthesize the same prekeratins as the colonic epithelium.

Animals↗
PubMed1995

Asymmetrical blastomere origin and spatial domains of dopamine and neuropeptide Y amacrine subtypes in Xenopus tadpole retina.

Amacrine cells are located almost exclusively in the inner nuclear layer (INL) of the retina, but they express a variety of neurotransmitters. To begin to elucidate the relative roles of the local environment and cell lineage in determining the different neurotransmitter subtypes of amacrine cells, we combined lineage tracing and immunocytochemical techniques to map the spatial distribution and clonal origin of dopamine (DA) and neuropeptide Y (NPY) amacrine cells in Xenopus tadpole retina. At the earliest period of neurotransmitter expression, both DA and NPY amacrine cells were distributed preferentially in center and intermediate annular regions, and in anterior and dorsal quadrants. Most of the DA and NPY cells first emerged as scattered cells and later as clusters (of 2 or more cells) that increased in number and size up to premetamorphic stages. These results suggest that DA and NPY amacrine subtypes may be influenced by environmental cues localized to specific regions of the retina. Lineage analysis showed that the percentage of DA or NPY amacrine cells produced by most blastomere progenitors is significantly different from that predicted by the number of cells in the retina produced by those blastomeres. Only two blastomeres produced over 90% of the DA amacrine cells and only four produced 97% of the NPY amacrine cells. Some retinal progenitors did not contribute at all to these two amacrine subtypes. There also is a marked asymmetry in the blastomere origin of DA and NPY amacrine cells. Two retinal progenitors produced significant numbers of NPY but very few DA amacrine cells. This analysis provides evidence that blastomere origin restricts the developmental choices of retinal progenitors.

Animals↗
PubMed1999

Intracellular fate mapping in a basal metazoan, the ctenophore Mnemiopsis leidyi, reveals the origins of mesoderm and the existence of indeterminate cell lineages.

Ctenophores are marine invertebrates that develop rapidly and directly into juvenile adults. They are likely to be the simplest metazoans possessing definitive muscle cells and are possibly the sister group to the Bilateria. All ctenophore embryos display a highly stereotyped, phylum-specific pattern of development in which every cell can be identified by its lineage history. We generated a cell lineage fate map for Mnemiopsis leidyi by injecting fluorescent lineage tracers into individual blastomeres up through the 60-cell stage. The adult ctenophore body plan is composed of four nearly identical quadrants organized along the oral-aboral axis. Each of the four quadrants is derived largely from one cell of the four-cell-stage embryo. At the eight-cell stage each quadrant contains a single E ("end") and M ("middle") blastomere. Subsequently, micromeres are formed first at the aboral pole and later at the oral pole. The ctene rows, apical organ, and tentacle apparatus are complex structures that are generated by both E and M blastomere lineages from all four quadrants. All muscle cells are derived from micromeres born at the oral pole of endomesodermal precursors (2M and 3E macromeres). While the development of the four quadrants is similar, diagonally opposed quadrants share more similarities than adjacent quadrants. Adult ctenophores possess two diagonally opposed endodermal anal canals that open at the base of the apical organ. These two structures are derived from the two diagonally opposed 2M/ macromeres. The two opposing 2M/ macromeres generated a unique set of circumpharyngeal muscle cells, but do not contribute to the anal canals. No other lineages displayed such diagonal asymmetries. Clones from each blastomere yielded regular, but not completely invariant patterns of descendents. Ectodermal descendents normally, but not always, remained within their corresponding quadrants. On the other hand, endodermal and mesodermal progeny dispersed throughout the body. The variability in the exact complements of adult structures, along with previously published cell deletion experiments, demonstrates that cell interactions are required for normal cell fate determination. Ctenophore embryos, like those of many bilaterian phyla (e.g., spiralians, nematodes, and echinoids), display a highly stereotyped cleavage program in which some, but not all, blastomeres are determined at the time of their birth. The results suggest that mesodermal tissues originally evolved from endoderm tissue.

Animals↗
PubMed1988

The somitic level of origin of embryonic chick hindlimb muscles.

Studies of avian chimeras made by transplanting groups of quail somites into chick embryos have consistently shown that the muscle cells of the hindlimb are derived from the adjacent somites, however, the pattern of cell distribution from individual somites to individual hindlimb muscles has not been characterized. I have mapped quail cell distribution in the chick hindlimb after single somite transplantation to determine if cells from an individual somite populate discrete limb muscle regions and if there is a spatial correspondence between a muscle's somitic level of origin and the known spinal cord position of its motoneuron pool. At stages 15-18 single chick somites or equivalent lengths of unsegmented somitic mesoderm adjacent to the prospective hindlimb region were replaced with the corresponding tissue from quail embryos. At stages 28-34, quail cell distribution was mapped within individual thigh muscles and shank muscle regions. A quail-specific antiserum and Feulgen staining were used to identify quail cells. Transplants from somite levels 26-33 each gave rise to consistent quail cell labeling in a unique subset of limb muscles. The anteroposterior positions of these subsets corresponded to that of the transplanted somitic tissue. For example, more anterior or anteromedial thigh muscles contained quail cells when more anterior somitic tissue had been transplanted. For the majority of thigh muscles studied and for shank muscle groups, there was also a clear correlation between somitic level of origin and motoneuron pool position. These data are compatible with the hypothesis that motoneurons and the muscle cells of their targets share axial position labels. The question of whether motoneurons from a specific spinal cord segment recognize and consequently innervate muscle cells derived from the same axial level during early axon outgrowth is addressed in the accompanying paper (C. Lance-Jones, 1988, Dev. Biol. 126, 408-419). Quail cell distribution was also mapped in chick embryos in which quail somites or unsegmented mesoderm had been placed 2-3 somites away from their position of origin. In all cases donor somitic tissues contributed to muscles in accord with their host position. These results indicate that muscle cell precursors within the somites are not specified to migrate to a predetermined target region.

Animals↗
PubMed2006

Direct thrombin inhibitors built on the azaphenylalanine scaffold provoke degranulation of mast cells.

The main structural feature of direct thrombin inhibitor LK-732 responsible for the appropriate interaction at the thrombin active site is a strong basic group. A possibility that a strong basic group of LK-732 might contribute to the mast cell degranulation effect and consequent reduction of tracheal air flow (TAF) and fall of mean arterial blood pressure (MAP) in rats was investigated in the present study. At doses up to 5 mg/kg (i.v.), LK-732 did not cause significant changes of TAF and MAP. At 7 mg/kg (i.v.), a sudden reduction of TAF and a fall of MAP was observed within 5 min after LK-732 administration (75% mortality, p = 0.007). A less basic direct thrombin inhibitor LK-658 (21 mg/kg, i.v.) did not significantly disturb TAF and MAP. A reduction of TAF and a fall of MAP caused by LK-732 (7 mg/kg, i.v.) was almost completely abolished in rats with degranulated mast cells (0% mortality, p = 0.008). LK-732 concentration-dependently degranulated rat peritoneal mast cells in vitro (pEC(50) = 1.92 +/- 0.05 muM). A structure-activity relationship (SAR) study revealed that the terminal basic groups attached to the aromatic ring are responsible for the mast cell degranulation effect. A good correlation was observed between mast cell degranulation and pK(b) of analogues of LK-732 (R(2) = 0.49), but not between mast cell degranulation and thrombin K(i) (R(2) = 0.23). LK-732-induced reduction of TAF, the fall of MAP and high mortality originate from LK-732-induced mast cell degranulation. As judged by the SAR study, this effect could be overcome by reducing the basicity of LK-732.

Animals↗
PubMed2004

Complementation of replication origin function in mouse embryonic stem cells by human DNA sequences.

A functional origin of replication was mapped to the transcriptional promoter and exon 1 of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in the mouse and human genomes. This origin was lost in mouse embryonic stem (ES) cells with a spontaneous deletion (approximately 36 kb) at the 5' end of the HPRT locus. Restoration of HPRT activity by homologous recombination with human/mouse chimeric sequences reconstituted replication origin activity in two independent ES cell lines. Quantitative PCR analyses of abundance of genetic markers in size-fractionated nascent DNA indicated that initiation of DNA replication coincided with the site of insertion in the mouse genome of the 335 bp of human DNA containing the HPRT exon 1 and a truncated promoter. The genetic information contained in the human sequence and surrounding mouse DNA was analyzed for cis-acting elements that might contribute to selection and functional activation of a mammalian origin of DNA replication.

Animals↗