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PubMed2003

MR microscopy of magnetically labeled neurospheres transplanted into the Lewis EAE rat brain.

Stem cell transplantation is being explored as a new paradigm for the treatment of demyelinating diseases. Magnetically labeled multipotential neural precursor cells were transplanted into the ventricles of rats with acute experimental allergic encephalomyelitis (EAE) and high-resolution (microscopic) MR images were obtained ex vivo. Migration patterns of live cells into periventricular white matter structures could be easily visualized, with a good correlation of the corresponding histopathology. The present results confirm that MR cell tracking can be used to guide the development of successful transplantation protocols.

Animals↗
PubMed2006

Use of a fluorescent internal protein standard to achieve quantitative two-dimensional gel electrophoresis.

2-DE is a powerful separation method for complex protein mixtures. However, large intergel variations in spot intensity limit its use for quantitative proteomics studies. To address this issue, we developed a fluorescent internal protein standard for use in 2-DE analysis. Protein samples are spiked with an Alexa-labeled internal standard (ALIS) prior to separation with 2-DE. Due to the high extinction coefficient of the Alexa-fluor, incorporation of 0.1% of total protein is sufficient to allow visualization of the internal standard yet low enough to avoid interference in subsequent quantification and identification steps. Following 2-DE, total proteins are visualized with fluorescent postelectrophoretic stains spectrally separated from ALIS. Four protein stains, Deep Purple, Sulforhodamine G, ruthenium II-tris(bathophenanthroline disulfonate) (RuTBS), and SYPRO Ruby, including improved purification and staining protocols for RuTBS and ten-fold dilutions of SYPRO Ruby were evaluated. All staining protocols were compatible with the ALIS method and had similar LODs (1-4 ng) and dynamic ranges (10(3)). ALIS is a powerful normalization method for quantitative 2-DE which avoids potential problems associated with dual spot migration patterns observed in the DIGE method. Furthermore, ALIS provides significantly improved normality in the distribution of spot abundance-variance compared to normalization through division by the total spot volume.

Animals↗
PubMedDate not supplied

Application of confocal laser scanning microscopy to analysis of H2O2-induced DNA damage in human cells.

Confocal laser scanning microscopy (CLSM) offers improved depth discrimination and spatial resolution to the analysis of biologic samples. We demonstrate in this paper that such technology is valuable in examining DNA single-strand breaks in human cells. The single-cell-gel (SCG) assay is a new technique for measuring DNA strand breaks in individual cells. Cells embedded in low-melting-point agarose are treated with varying concentrations of hydrogen peroxide to induce DNA strand breaks. Following cell lysis and alkaline electrophoresis, which enables single-stranded break detection, analysis of the resulting "comets" provides an accurate method of comparing changes in DNA migration patterns, which have been shown to reflect the DNA damage levels. A statistically significant difference (p < 0.01) in single-stranded DNA damage levels was detected in cells exposed to hydrogen peroxide concentrations as low as 10 nm for 2 min. LSM analysis of the SCG technique allows rapid, sensitive, and reproducible quantitation of single-stranded breaks of cellular DNA.

Cell Line↗
PubMed2000

Identification, characterization, and immunolocalization of a nucleoside triphosphate diphosphohydrolase in pig liver.

Different isoforms of nucleoside triphosphate diphosphohydrolases (NTPDases; EC 3.6.1.5), also identified as ATP diphosphohydrolases, have been previously described in mammalian tissues. We report here the biochemical characterization of NTPDases in the pig liver. Optimum pH of catalysis is more acidic for this enzyme than for NTPDases (neutral or alkaline pH) found in other mammalian tissues. It is less sensitive to bile salts than the bovine spleen NTPDase. Calculated Km values for ATP and ADP (31 and 21 microM, respectively) are slightly higher than those reported for the latter enzyme. Electrophoretograms of these enzymes also show different migration patterns. Western blots with Ringo, an antibody that recognizes the different isoforms of mammalian NTPDases, show a small but reproducible difference in estimated molecular masses (75 kDa for liver vs 78 kDa for spleen NTPDase). A second antibody, generated against a different sequence of NTPDase I, does not recognize the liver enzyme, thereby indicating some differences in primary structure. Immunolocalization produced a strong signal on hepatocytes, epithelial cells of the bile duct system, and vascular cells. Immunoreactivity was variable among hepatocytes of different lobules and among hepatocytes within a given lobule. In general, those located in the perilobular zone were more reactive than those located in the central zone and in the periphery of the centrolobular vein.

Acid Anhydride Hydrolases↗
PubMed1993

Molecular mechanisms of the increased glucose transport in transformed FRTL-5 rat thyroid cells.

We investigated the molecular mechanisms involved in the glucose carrier (Glut 1) regulation in FRTL-5 cells and two derived transformed clones (SRC and Ki-Mol cells). When compared to the wild-type strain, SRC and Ki-Mol cells showed an increase in both glucose consumption and uptake (about 60 fold), associated with 6-8 fold higher Glut 1 mRNA levels. Transcriptional studies revealed a 2- to 3 fold increased activation of the gene in the transformed cells, suggesting that transcription alone cannot fully account for the higher Glut 1 gene expression. Western blot studies showed an increase of the Glut 1 protein in SRC and Ki-Mol cells, associated with a different gel migration pattern and a disparate distribution rate between the plasma membrane and the microsomal fraction. These data indicate that the higher rate of glucose uptake observed in SRC and Ki-Mol cells is associated to an increase in Glut 1 gene expression, and that also changes in the subcellular distribution and probably in the structure of Glut 1 protein are present.

Animals↗
PubMed1997

Characterization of recombinant bovine conglutinin expressed in a mammalian cell.

We describe here the successful expression of recombinant bovine conglutinin in CHO cells as well as its physical and biological characteristics. Geneticin-resistant transformants harboring bovine conglutinin cDNA in the expression vector pNOW/CMV-A were screened by Western blot analysis for secretion into media of recombinant conglutinin. A four-day amplification of the transgene with increasing concentrations of methotrexate resulted in a dose-dependent increase in the production of recombinant conglutinin to a final concentration of 18.6 microg/ml of media. Recombinant conglutinin purified from this media by affinity column chromatography on mannan-agarose had a migration pattern similar to that of native conglutinin on polyacrylamide gel electrophoresis under reducing, nonreducing, and native conditions. The recombinant conglutinin exhibited sugar binding, conglutination, hemagglutination inhibition, and neutralization of influenza A virus, activities engaged in by the native conglutinin. This is the first report describing a high level of expression of a serum cruciform collectin with the full range of biological activity.

Animals↗
PubMed1993

Four new mutations in the ornithine transcarbamylase gene.

We characterized four new mutations in the ornithine transcarbamylase (OTC) gene in three male infants who died from acute neonatal hyperammonemia and one male infant with late onset disease. OTC enzymatic activity was undetectable in the livers of the three neonates, whereas residual enzymatic activity was present in the fourth patient. All 10 exons of the OTC gene were amplified by the polymerase chain reaction (PCR) from genomic DNA of the four patients. The amplified DNA was screened for abnormal gel electrophoretic migration patterns via single-strand conformational polymorphism (SSCP). One patient showed an abnormal SSCP pattern of exon 8 and exon 9, a second patient had an abnormal exon 6, a third had an abnormal exon 9, and the fourth patient revealed an abnormal exon 3. Sequencing of the abnormal exons revealed that the first patient had a deleterious mutation in exon 9 consisting of a G-->T transversion in codon 310 causing a Glu-->stop coding change. The abnormal exon 8 of this patient contained a common polymorphism consisting of an A-->G transition in codon 270 resulting in Gln-->Arg code change. The abnormal exon 6 of the second patient contained an A-->G transition in codon 183 causing a Tyr-->Cys change. Exon 9 of the third patient contained a deletion of a thymine nucleotide (base 882) resulting in a shift of the reading frame and a code for premature termination 28 codons downstream. The fourth patient with a "milder" clinical presentation had an A-->T transversion in exon 3 (codon 88) causing a Lys-->Asn change.

Ammonia↗
PubMed1994

Localization of prothymocytes from wild-type and viable motheaten mice following intravenous injection into irradiated adoptive recipients.

Bone marrow cells from viable motheaten (me(v)) mutant mice fail to repopulate the thymus of irradiated recipients upon i.v. adoptive transfer. This defect can be corrected by the addition of wild-type marrow to the me(v)/me(v) marrow prior to i.v. injection. The present study focused on the nature of this apparent defect in me(v)/me(v) prothymocyte activity. We established an adoptive transfer assay system that facilitated the investigation of the migration patterns of wild-type and me(v)/me(v) marrow prothymocytes in adoptive recipients. Wild-type prothymocytes were found to seed the thymus of recipients as early as 1 day after i.v. injection. This early wave of prothymocyte seeding peaked at 4 days and diminished to undetectable levels at 6 days postinjection. After an interval of no detectable wild-type precursor activity in the thymus of primary recipients, a second wave of activity was observed on Day 15. This second wave resulted in permanent engraftment and persisted throughout the observation period. In contrast, only one initial wave of me(v)/me(v) prothymocyte seeding occurred in the recipient thymus. Detection of mev/mev precursors in the recipient's thymus was dependent upon the coinjection of wild-type marrow cells. These results suggest that me(v)/me(v) prothymocytes initially seed to the thymus of adoptive recipients, but fail to proliferate and/or differentiate due to a defect in marrow-derived intrathymic accessory cell activity.

Animals↗
PubMed1994

The contribution of both forebrain and midbrain crest cells to the mesenchyme in the frontonasal mass of mouse embryos.

Migration of cranial neural crest cells is a crucial event in the formation of facial organs such as the frontonasal mass and branchial arches. However, the source of the populating crest cells that occupy the frontonasal mass remains unclear in mammalian embryos. To elucidate this, we performed focal DiI injections at various sites in the prosencephalon (forebrain, including the future telencephalon and diencephalon), mesencephalon (midbrain), and the anterior part of the rhombencephalon (hindbrain) separated posteriorly by the preotic sulus (i.e., rhombomere A; future rhombomere 1 and 2) of cultured mouse embryos from the 3- to 10-somite stage. Results directly revealed that during these stages the lateral edge of the prosencephalon produced crest cells which migrated to the frontonasal mass. On the other hand, labeled cells at the anterior neural ridge in the prosencephalon contributed mainly to the head epithelium, including the nasal placode, Rathke's pouch, and oral epithelium. As for the crest cells of the mesencephalon and rhombomere A, their destinations were significantly dependent on the injection site and somite stage. At the 3- to 4-somite stage, the crest cells emigrating from both the mesencephalon and rhombomere A migrated to the first branchial arch. Moreover, the mesencephalic region, but never rhombomere A, produced another group of crest cells that migrated to the frontonasal mass. In the 5- to 10-somite stage, the destinations of late-emigrating crest cells were restricted depending on their premigratory positions, i.e., the region producing crest cells migrating toward the frontonasal mass was restricted to the anterior portion of the mesencephalon, and the crest cells from the posterior portion of the mesencephalon primarily migrated to the first branchial arch, while those from the rhombomere A predominantly migrated to the trigeminal ganglion. Migration toward the frontonasal mass from the mesencephalon ceased at the earliest in the 7-somite stage, followed by termination of mesencephalic and rhombencephalic crest cell migration toward the first branchial arch at the 8-somite stage, whereas the contribution from rhombomere A to the trigeminal ganglion continued even at the 10-somite stage. This behavior suggests that both the prosencephalic and mesencephalic crest contribute to the mesenchymal cells in the frontonasal mass and also that the migration patterns of crest cells released from the prosencephalon, mesencephalon, and rhombencephalon depend on their axial level and developmental stage at initial emigration.

Animals↗
PubMed1998

GDNF and ET-3 differentially modulate the numbers of avian enteric neural crest cells and enteric neurons in vitro.

Vagal (hindbrain) neural crest cells migrate rostrocaudally in the gut to establish the enteric nervous system. Glial-derived neurotrophic factor (GDNF) and its receptor(s), and endothelin-3 (ET-3) and its receptor, are crucial for enteric nervous system development. Mutations interrupting either of these signaling pathways cause aganglionosis in the gut, termed Hirschsprung's disease in humans. However, the precise functions of GDNF and ET-3 in enteric neurogenesis are still unknown. We isolated precursor cells of the enteric nervous system from the vagal level neural crest of E1.7 quail embryos prior to entry into the gut and from the developing midgut at stages corresponding to migrating (E4.7) and longer resident differentiating cells (E7) using HNK-1 immunoaffinity and magnetic beads. These cells were tested for their response to GDNF and ET-3 in culture. ET-3 and GDNF had little effect in vitro on the growth, survival, migration, or neurogenesis of E1.7 vagal neural crest cells. In contrast, GDNF increased the proliferation rate and numbers of enteric neural precursors isolated from the E4.7 and E7 gut. Also, many more neurons and neurites developed in cultures treated with GDNF, disproportionately greater than the effect on cell numbers. At high cell density and in the presence of serum, ET-3, and GDNF had an additive effect on proliferation of neuron precursor cells. In defined medium, or low cell density, ET-3 reduced cell proliferation, overriding the proliferative effect of GDNF. Regardless of the culture condition, the stimulatory effect of GDNF on neuron numbers was strikingly diminished by the simultaneous presence of ET-3. We propose first that GDNF promotes the proliferation in the migratory enteric neural precursor cell population once the cells have entered the gut and is especially crucial for the differentiation of these cells into nonmigrating, nonproliferating enteric neurons. Second, we suggest that ET-3 modulates the action of GDNF, inhibiting neuronal differentiation to maintain the precursor cell pool, so ensuring sufficient population numbers to construct the entire enteric nervous system. Third, we suggest that generalized defects in enteric neural precursor cell numbers and differentiation due to mutations in the ET-3 and GDNF systems are converted to distal gut neural deficiencies by the rostrocaudal migration pattern of the precursors. Fourth, we suggest that additional factors such as those found in serum and produced by the enteric neural cells themselves are likely also to be involved in enteric nervous system development and consequently in Hirschsprung's disease.

Animals↗
PubMed1997

Electrophoretic karyotype of clinical isolates of Paracoccidioides brasiliensis.

Chromosomal DNA molecules were obtained from eight clinical isolates of Paracoccidioides brasiliensis during the early staged of the yeast to mycelial transformation process and were separated by contour-clamped homogeneous electric field gel electrophoresis. In seven of the isolates, it was possible to separate five bands, albeit with two different migration patterns (A and B). In the remaining isolate only four bands were observed (pattern C). The isolated chromosomes showed molecular sizes ranging from 3.2 to 10 Mb according to Schizosaccharomyces pombe molecular size standards. The three different patterns observed had bands of 3.2, 3.8, 4.1, 5.2, 6.7, 7.2, 8.8, and 10 Mb. These data imply that at least 30 Mb of the genome are organized as chromosomal macromolecules. Due to the fact that P. brasiliensis spheroplasts are difficult to obtain, technical details are provided.

Chromosomes, Fungal↗
PubMed1999

Identification, chromosomal assignment, and expression analysis of the human homeodomain-containing gene Orthopedia (OTP).

Homeodomain (HD) genes are helix-turn-helix transcription factors that play key roles in the specification of cell fates. In the central nervous system (CNS), HD genes not only position cells along an axis, but also specify cell migration patterns and may influence axonal connectivity. In an effort to identify novel HD genes involved in the development of the human CNS, we have cloned, characterized, and mapped the human homologue of the murine HD gene Orthopedia (Otp), whose product is found in multiple cell groups within the mouse hypothalamus, amygdala, and brain stem. Human cDNA and genomic libraries were screened with probes derived from mouse Otp sequences to find the human homologue, OTP. The deduced amino acid sequence of the open reading frame of the human cDNA is 99% homologous to mouse Otp and demonstrates a high degree of conservation when compared to sea urchin and Drosophila. OTP was mapped to human chromosome 5q13.3 using radiation hybrid panel mapping and fluorescence in situ hybridization. Flanking markers were identified from YAC clones containing OTP. A single putative OTP gene product was found in 17-week human fetal brain tissue by Western blot analysis using a novel polyclonal antibody raised against a conserved 13-amino-acid sequence at the C-terminus of the OTP protein. Expression in the developing human hypothalamus was confirmed by immunohistochemistry.

Amino Acid Sequence↗
PubMed1998

Mutations in T cell receptor zeta chain mRNA of peripheral T cells from systemic lupus erythematosus patients.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of unknown aetiology. Although it has been reported that T cells might be responsible for the pathogenesis of SLE, it remains unclear whether immune aberrations of SLE T cells are the primary event in this pathological process. We have recently reported that tyrosine phosphorylation and expression of the T cell receptor zeta chain (TCR zeta) was significantly decreased in SLE T cells and that two SLE patients exhibited a 36 bp, exon 7 deletion of the TCR zeta mRNA. To investigate further common mutations in TCR zeta mRNA among SLE patients, mRNA was isolated from the peripheral blood T cells of two normal controls, two systemic sclerosis (SSc) patients, and eight SLE patients. TCR zeta cDNA was amplified by RT-PCR. Five out of the eight SLE patients exhibited abnormal migration patterns of the TCR zeta cDNA in PCR single stranded conformational polymorphism analysis. PCR products were ligated into pUC18 and five clones obtained were sequenced. Analysis of the nucleotide sequences revealed that all of the five pUC18 clones from the normal controls and SSc patients had the normal nucleotide sequence, whereas all eight SLE patients had mutations in TCR zeta cDNA accompanied by predicted amino acid substitutions. Mutations found in six of these patients corresponded to those of the third immunoreceptor tyrosine-based activation motif (ITAM) domain or the GTP/GDP binding site in TCR zetaThus, these mutations in TCR zeta mRNA could be responsible for the decreased expression of the TCR zeta protein in SLE T cells.

Amino Acid Substitution↗
PubMed1999

Identification of two single nucleotide polymorphisms in exon 8 of PAX2.

We estimate the allele frequencies of two single nucleotide polymorphisms (1410 C --> T) and (1521 A --> C) in the coding region of PAX2. The coding region single nucleotide polymorphisms (cSNPs) were identified by sequencing of amplimers of PAX2 exon 8 exhibiting variant migration patterns in the course of genomic DNA mutation screening from patients with renal-coloboma syndrome. Allele frequencies of the two polymorphisms were 0.94 for 1410C and 0.72 for 1521A. Cosegregation analyses of both alleles suggest that they are each in Hardy-Weinberg equilibrium and jointly in linkage equilibrium and may represent ancient polymorphisms. Characterization of PAX2 exon 8 cSNPs will serve as useful tools for mapping at the PAX2 locus.

Abnormalities, Multiple↗
PubMed1995

The quaternary structure of the adenovirus 12 early region 1B 54K protein.

The quaternary structure of the adenovirus early region 1B 54K protein has been examined under denaturing and nondenaturing conditions. In the presence of SDS the protein has a strong tendency to form disulfide-linked high-molecular-weight polymers. Under nondenaturing, but reducing, conditions the in vitro-translated 54K polypeptide forms oligomers (probably tetramers) of molecular weight approximately 2 x 10(5). After subcellular fractionation of Ad12 early region 1-transformed cells, the 54K E1B protein present in the cytoplasm had a molecular weight similar to that determined for the in vitro-translated material. However, two populations of the viral protein could be distinguished in the nucleus-one of a size similar to that seen in the cytoplasm and the other of appreciably higher molecular weight (approximately 2 x 10(6)). No difference in migration pattern was observed after treatment of the nuclear extract with DNase I or RNase. A proportion of the Ad12 E1B 54K protein in both the high- and the low-molecular-weight populations in the nucleus was found to form a complex with p53, and it is therefore concluded that the very high molecular weight derives from interaction with an, as yet, unidentified component.

Adenovirus E1B Proteins↗
PubMed1997

Expression, purification, and characterization of a soluble form of human cytomegalovirus glycoprotein B.

The human cytomegalovirus glycoprotein B gene (gB; gpUL55) was truncated at amino acid 692 and recombined into Autographa californica nuclear polyhedrosis virus (baculovirus). Infection of insect cells with the recombinant baculovirus resulted in high-level expression and secretion of the truncated gB protein (gB-S) into the culture medium. Purification of gB-S by monoclonal antibody affinity chromatography yielded a protein of ca. 200 kDa. Characterization of the 200-kDa purification product indicated that the recombinant gB protein retained many structural and functional features of the viral gB. Comparison of electrophoretic migration patterns in reduced versus nonreduced protein samples and immune blotting analysis with antibodies specific for the amino or carboxy-terminus of gB demonstrated that the recombinant protein was composed of disulfide linked 69 kDa amino terminal and 35-kDa carboxy-terminal fragments. In addition, recognition of the 200-kDa gB-S by a conformational-dependent, oligomer-specific monoclonal antibody suggested that gB-S was properly folded and dimeric. Like the viral gB, gB-S had heparin binding ability. One heparin binding site was found to reside within the 35-kDa carboxy-terminal fragment (aa 492-692). Heparin binding was abolished when gB-S was denatured. These data suggest that gB contains a novel heparin binding motif that is at least partially conformational dependent.

Cytomegalovirus↗
PubMed1982

Gut and bronchus associated lymphoid tissue: an overview.

The mucosal lymphoid aggregates in lung and gut contain precursor cells destined to seed mucosal tissues with IgA containing cells. It is likely that both tissues also are sources of both IgE B cell precursors as well as the cells responsible for their regulation (38). Although the traffic of B cells is relatively well understood from the standpoint of IgA, the factors responsible for localization in mucosal tissue are at best unclear. Furthermore, little is known about the migration patterns of helper or suppressor cells of any kind and these may have, if derived from mucosal tissue, a preferential site of action in the mucosa. The mucosal mast cell may well be a cell of different lineage than the mast cell from the peritoneal cavity and may itself have a mucosal localization pattern. Much more work needs to be done to render some of these speculations onto a better factual base, and to harness these systems in provision of better approaches to vaccination and control of disease.

Animals↗
PubMed1991

Orientation in birds. Satellite tracking: a new method in orientation research.

The study of bird migration by satellite tracking began in the 1980s, after transmitters had become sufficiently small to allow the monitoring of larger migrating species. The initial studies in this direction were all successful with respect to establishing this method (Section 2) of elucidating new migration patterns and various aspects of orientation. These pilot studies are summarized in Section 3, Table 1, Figures 1-4. We believe that studies on avian orientation and navigation will, in general, greatly benefit from satellite tracking if some prerequisites are fulfilled and adequate experiments planned (Section 4). In future studies, smaller transmitters with increased life expectancies are needed, and possible effects of these transmitters on bird behaviour must be examined. Considerable improvement of satellite tracking in avian orientation research is to be expected, along with the development of a new generation of satellites allowing communication between satellites and transmitters (Sections 4, 5).

Animals↗