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Biomedical subjects

L C Smith

Publications and source records attributed to L C Smith.

At least 91 records · Page 5Linked to original sources

Gene therapy in heart disease.

As the technology for gene therapy develops in vitro and in vivo in animal models, it is becoming clear that the three principal approaches--recombinant retroviruses, recombinant adenovirus, and direct DNA delivery--will ultimately have applications in specific therapeutic situations that take full advantage of the unique features of the specific delivery system: low level persistent expression after ex vivo recombinant retroviral therapy, high level transient expression after in vivo recombinant adenoviral therapy, or moderate level transient expression after in vivo administration of a synthetic DNA complex, which in principle could be repeated as desired.

Animals↗

Viability of cloned bovine embryos after one or two cycles of nuclear transfer and in vitro culture.

We described an exclusively in vitro procedure for cloning and recloning bovine embryos. Embryos obtained by IVM/IVF/IVC developed to the morula stage were used as blastomere donors in cunjunction with IVM recipient oocytes. Reconstructed embryos were developed in vitro in co-culture using bovine oviductal epithelial cells. The resulting morulae were used as donors for recloning under the same experimental conditions. No significant difference was observed between cloning and recloning in terms of development (rates of blastocysts: 12.9 versus 14.9%), in the number of nuclei per blastocyst (63.8 versus 49.1), or in pregnancy rates (35.7 versus 33.3%). The high variability observed between replicates and the correlation between results in first and second cycle nuclear transfer may suggest an inherant potential of individual donor embryos to support development by cloning.

Journal Article↗

Development of immortalized endometrial epithelial and stromal cell lines from the mink (Mustela vison) uterus and their effects on the survival in vitro of mink blastocysts in obligate diapause.

Mink endometrial cell lines were established by stable transfection of a plasmid vector encoding the SV40 large T antigen driven by the human beta-actin promoter. A second plasmid vector, pSV2neo, was employed for selection of transfected cells. Specificity and homogeneity of consequent cell lines were evaluated by immunocytochemistry employing antibodies against cytokeratin, desmin, and vimentin. Cytokeratin was found exclusively in epithelial cells, whereas vimentin appeared primarily in stromal cells. Neither cell line showed detectable desmin activity. These cell lines along with Buffalo rat liver (BRL) cells were employed in coculture with mink embryos in obligate diapause. Mink stromal and BRL cell lines were most effective in enhancing embryo survival in vitro. The percentages of cocultured embryos that survived for 72 h or more were 65% with epithelial cells, 75% with stromal cells, 68% with the combination of stromal and epithelial cells, and 93% with BRL cells. Only 23% of the embryos cultured without cells survived beyond 48 h. Embryo growth was also observed; some embryos in coculture showed trophoblastic outgrowth and adhesion to the cell surfaces. These results demonstrate that mink embryos in obligate delay can survive and develop in culture and that coculture with uterine or BRL cells increases the length and frequency of survival.

Alkaline Phosphatase↗

Eigenanalysis of DAPI-stained chromosomes: tools and strategies toward computer-assisted analysis of FISH experiments.

The fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) is widely used as a chromosome counterstain in fluorescence in situ hybridization (FISH) studies. It produces a Q-banding pattern that allows for both chromosome identification and the assignment of molecular probes to specific chromosome bands. Using a statistical procedure based on eigenanalysis, we have extracted features from digital images of DAPI-stained chromosomes and constructed prototypes of each of the 24 human chromosomes. The features of these prototypes are directly proportional, in intensity profile and band location, to those of real chromosomes. The prototype's intensity profile can be translated into cytogenetic bands to provide a computer-based strategy for chromosome mapping and analysis amenable to automation. Data presented here were obtained using images from the 24 human chromosomes and mouse X chromosome. Moreover, the same procedure is general and can be used for the analysis of chromosomes from other species, as well as with banding techniques other than those using DAPI.

Animals↗

Mapping segmental imbalances using comparative genomic hybridization and eigenanalysis.

We have tested a new approach to comparative genomic hybridization (CGH) analysis using digital ratio images and eigenanalysis, which allows the recognition of consistent patterns along the chromosomes and discards random (background noise) patterns. We have performed test experiments using genomic DNAs from a patient with a duplication, another with a deletion of a chromosome segment, and a prostate cancer biopsy. Image ratio analysis was performed, and ratio images of the relevant chromosome were subjected to eigenanalysis. The results showed a high-contrast enhancement of the regions corresponding to the unbalanced genomic segment, with clearly defined limits between normal and abnormal fluorescence ratios. The combination of digital ratio images and eigenanalysis allowed the precise mapping of unbalanced regions consistent with other methods of analysis. Because there is no limit to the number of chromosomes that can be analyzed at any one time, the method has the potential of increasing the sensitivity of CGH by reducing the noise component.

Cell Line↗

Efficient gene delivery and expression in mammalian cells using DNA coupled with perfringolysin O.

Current non-viral DNA vectors for gene therapy are limited by low cellular transfection efficiencies and low levels of gene expression due to inefficient endosomal DNA release. We have used perfringolysin O (PFO), a membrane active bacterial protein, to deliver DNA into cells. PFO belongs to the so-called sulphydryl-activated family of membrane active bacterial proteins, which have been used to deliver small molecules and proteins into cells. PFO was incorporated into DNA complexes through a biotin-streptavidin bridge and the DNA-PFO complexes were used to deliver the Escherichia coli beta-galactosidase gene into cells. High levels of gene expression were achieved in murine sol 8 myoblast cells using these DNA-PFO complexes. The level of gene expression correlated well with the content of PFO in the complexes. Under optimal conditions, 15-20% of the cells were stained blue with X-gal. Furthermore, the expression was independent of a receptor ligand. Thus, membrane active bacterial proteins may be an important tool for the future development of non-viral DNA delivery systems for gene therapy.

Animals↗

A nonexchangeable apolipoprotein E peptide that mediates binding to the low density lipoprotein receptor.

ApoE is a 34-kDa apoprotein that mediates lipoprotein binding to the low density lipoprotein (LDL) receptor and to the LDL receptor-related protein. Receptor binding is mediated by a highly basic, alpha-helical sequence of approximately 15 amino acids that interacts with cysteine-rich repeat regions of the receptor. To determine the relationship between the receptor binding and lipid associating properties of apoE, we have synthesized a series of apoE peptides containing all (residues 129-169) or part (residues 139-169, 144-169, and 148-169) of the receptor-binding domain. The lipophilicity of these peptides was increased by modification of their N termini by acylation with either palmitic acid (C16-apoE peptide) or the N,N-distearyl derivative of glycine (diC18-Gly-apoE peptide). The unmodified peptides demonstrated low affinity for lipid surfaces (Kd > 10(-5) M) and moderate alpha-helicity in the presence of lipid (40%) and had no effect on LDL uptake by fibroblasts. N-Palmitoyl peptides had increased affinity for lipid (Kd approximately 10(-6) M) and increased alpha-helicity (55%) in the presence of lipid. The addition of the C16-apoE-(129-169)-peptide to 125I-LDL enhanced its uptake and degradation by fibroblasts 8-10-fold; however, < 50% of the degradation was mediated by the LDL receptor. By contrast, the diC18-Gly-apoE-(129-169)-peptide was essentially nonexchangeable (Kd < or = 10(-9) M) and highly helical (78%) in the presence of lipid. The addition of the diC18-Gly-apoE-(129-169)-peptide to 125I-LDL enhanced the specific uptake and degradation of LDL by both LDL receptor-mediated and non-LDL receptor-mediated mechanisms. Uptake and degradation of methylated LDL containing diC18-Gly-apoE-(129-169) revealed that the lipoprotein-bound peptide is the active agent. In agreement with this finding, a mutant diC18-Gly-apoE peptide (Arg142-->Gln) was much less effective than the wild-type peptide in potentiating binding, uptake, and degradation of 125I-LDL. Complexes of diC18-Gly-apoE-(129-169), apoA-I, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine containing four to six copies of the peptide/particle displayed an affinity for the LDL receptor similar to that of apoE-L-alpha-dimyristoylphosphatidylcholine discs containing four copies of apoE.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

The echinoderm immune system. Characters shared with vertebrate immune systems and characters arising later in deuterostome phylogeny.

In summary, the characters of the echinoderm immune system that we review here can be considered to illuminate the baseline nonadaptive immune systems that were our original deuterostome heritage. We still retain--and greatly rely upon--similarly functioning, nonadaptive cellular defense systems. It is worth stressing that sea urchins are long lived, normally healthy animals that display remarkable abilities to heal wounds and combat major infections. From an external point of view, their immune systems obviously work very well. Thus, their cellular defense systems are extremely sensitive, and they respond rapidly to minor perturbations, all without any specific adaptive capabilities. These systems probably function through the transduction of signals conveying information on injury and infection, just as do the equivalent systems that underlie and back up our own adaptive immune systems, and that provide the initial series of defenses against pathogenic invasions. Many extremely interesting questions remain regarding the evolution of the deuterostome immune response. Are the echinoderm and tunicate systems the same, or have the protochordates augmented the basic phagocyte system with an as yet unidentified chordate-like character? Do the jawless fishes produce Igs that would make them similar to the sharks, or are they vertebrates without an Ig system that essentially rely on an invertebrate-like, nonspecific, activated phagocyte type of immune system? How do sharks regulate their immune system without T cells and MHC class I? How do they avoid producing autoantibodies? Future research will not only answer these questions, but those answers will also be enlightening with regard to the origins of the mammalian immune system in which ancient functions and subsystems remain.

Animals↗

The sea urchin profilin gene is specifically expressed in mesenchyme cells during gastrulation.

Eggs and embryos of the purple sea urchin (Strongylocentrotus purpuratus) contain profilin that is partly supplied from maternal sources and partly produced by the gastrula. The maternal profilin protein content is about 13 microM and it persists in the embryo at least through gastrulation. Transcript quantitation from probe excess titrations show that very few profilin gene transcripts are present in the embryo during cleavage, but that they increase at the onset of gastrulation. By in situ hybridization, the newly synthesized profilin transcripts are localized in mesenchyme cells. Profilin gene expression increases when mesenchyme cells initiate migration and filopodial extension and retraction. We show that there are three isoforms of maternal profilin protein produced from the single copy gene during oogenesis. However, the blastula stage embryo only produces the major isoform, whereas the acidic isoform is produced in the early stages of gastrulation and the basic isoform appears by the end of gastrulation. Based on transcript prevalence and protein production rates, our calculations indicate that the amount of new protein produced in the mesenchyme cells in 12 hr is at maximum < 2% of that supplied from maternal sources. Because of the large amount of maternally supplied profilin present in the egg and embryo, we suggest that it may be used in the cytokinetic processes of cleavage. Alternatively, because of the small amount of embryonically produced profilin, we suggest that it may function in the cytoskeletal shape changes required for filopodial extension and motility in the mesenchyme cells during gastrulation.

Animals↗

Ligand-dependent stimulation of introduced mammalian brain receptors alters spicule symmetry and other morphogenetic events in sea urchin embryos.

Zygotes of S. purpuratus were injected with synthetic mRNAs encoding rodent brain neurotransmitter receptors, and specific developmental phenotypes were produced on addition to the sea water of the respective ligands. Most of these experiments were carried out with a mouse serotonin receptor (5HT-R) mRNA, though exactly comparable results were obtained with a rat muscarinic acetylcholine receptor (MAChR) mRNA; these receptors are expected to couple to the same endogenous signal transduction system. We show by whole mount in situ hybridization that the injected mRNAs diffuse to all of the early blastomeres, and that they are translated in vivo. Three specific phenotypes were reproducibly observed. The most severe, occurring at highest levels of injected mRNA, was a cleavage arrest phenotype in which no overtly differentiated cells ever appear, though the embryos remain alive for at least 72 h. A gastrular arrest (GA) phenotype is generated in appreciable fractions of embryos developing from eggs injected with lower levels of mRNA. In GA embryos the blastocoel is filled with disorganized mesenchyme cells, including pigment cells and skeletogenic cells; there is no archenteron; and the entire ectoderm expresses an oral ectoderm cell surface marker. The least severe phenotype that we recognized displays an altered arrangement of spiculogenic foci (RSE phenotype), generating a ring of extra spicules that are properly positioned with respect to the animal/vegetal axis, but that lack any reference to the oral/aboral axis. However, use of cytological and molecular markers demonstrates that RSE embryos retain normal spatial patterns of aboral and oral ectoderm. They develop a fully formed archenteron, but fail to form either a stomodaeum or a ciliated band. RSE embryos can be produced in embryos expressing the 5HT-R by exposure to serotonin, beginning as late as 12 h postfertilization (pf). All of the morphogenetic processes affected in RSE embryos depend in normal embryos on intercellular interactions occurring at the blastula-gastrula stages of development.

Animals↗

Recurrent infection due to Legionella pneumophila in a patient with AIDS.

We report a patient with AIDS who presented with community-acquired cavitary Legionella pneumophila pneumonia. The patient recovered after an extended course of treatment with macrolide antibiotics. He returned to the hospital 4 months later with a febrile illness. Chest radiograms appeared normal. Cultures of blood yielded L. pneumophila. The isolate from blood was indistinguishable from the isolate from sputum taken during the first infection, as shown by restriction-endonuclease analysis and pulsed-field gel electrophoresis. These data suggest that the second infection represented reactivation of a persistent focus of infection that was not apparent when the patient had pneumonia.

AIDS-Related Opportunistic Infections↗

Retinol-binding protein: immunolocalization of protein and abundance of messenger ribonucleic acid in conceptus and maternal tissues during pregnancy in pigs.

Retinol-binding protein (RBP) is a major secretory product of liver as well as uterine endometrium and periimplantation conceptuses of pigs. The present study examined distribution of RBP protein and abundance of RBP mRNA in pig maternal and conceptus tissues throughout pregnancy. A porcine liver RBP cDNA clone was isolated and used as probe in these studies. Northern blot analysis detected a 1.0-1.1-kb transcript in conceptus, trophoblast, yolk sac, placenta, endometrium, myometrium, oviduct, and numerous fetal tissues. Slot blot analyses of RBP mRNA abundance in endometrium, myometrium, conceptus, trophoblast, chorioallantoic placenta, and embryo/fetus indicated differential RBP gene expression in each tissue. Immunocytochemical localization studies indicated the presence of immunoreactive RBP in endometrial surface and glandular epithelium, circular and longitudinal muscle of myometrium, parenchymal cells of fetal liver, and proximal convoluted tubules of fetal kidney. Results suggest an integrated system of RBP gene expression and protein secretion that allows for transport of retinol from the uterine endometrium to the periimplantation conceptus during early pregnancy and to the fetal-placental unit throughout pregnancy in pigs.

Allantois↗

Three-dimensional laser-scanning confocal microscopy of in situ hybridization in the skin.

In situ hybridization is an important tool in molecular and developmental biology to detect specific nucleic acid sequences (either mRNA or DNA) within cells. This technique is especially applicable to tissue sections since it provides information about the spatial distribution of DNA or mRNA sequences. However, previous studies utilizing in situ hybridization in the skin were hampered by a high degree of nonspecific background, which has made interpretation of the results difficult. In this paper, we demonstrate how refinements in in situ hybridization techniques, combined with laser-scanning confocal microscopy, significantly reduce nonspecific background and produce improved resolution of in situ hybridization in skin specimens. The sensitive detection method of laser-scanning confocal microscopy allows three-dimensional localization of S35 radioactive-labeled riboprobes within the emulsion of specimens, which is not possible with conventional bright or dark field light microscopy.

Dendritic Cells↗

Control of cleavage and further development in vitro in reconstituted two-cell mouse embryos.

Nuclear-cytoplasmic interactions during the second cell cycle of mouse embryos were examined by assessing the timing of cleavage in reconstituted two-cell embryos and their ability to develop further to the blastocyst stage in vitro. Nuclear transplantations were performed either within or across the cell cycle and at different stages of the cell cycle to assess the effect of 'asynchrony' on development. In most cases, cleavage occurred at an intermediate time between nuclear and cytoplasmic controls indicating an interaction in their mechanisms for controlling the timing of cleavage. Early nuclei extended the cleavage timing of late cytoplasm for a short period, possibly to allow for completion of DNA synthesis, while early cytoplasm delayed the expected cleavage time of late nuclei, possibly to enable proper maturation of cytoplasmic components. However, a block of cleavage was observed in most across cell cycle transplantations and also when fusing early two-cell karyoplasts to enucleated late two-cell blastomeres. It is suggested that this incompatibility is caused by major changes in the transcriptional status of donor and recipient cells. Although the development of reconstituted embryos to the blastocyst stage was clearly affected by cell cycle 'asynchrony' in within cell cycle transplantations, independent effects of cytoplast stage and, to a lesser extent, of karyoplast cell cycle stage were predominant in transplantations using eight-cell karyoplasts.

Animals↗

Folate receptor mediated DNA delivery into tumor cells: potosomal disruption results in enhanced gene expression.

We have used a particular folate receptor, which is overexpressed in tumor cells, for targeted DNA delivery into these cell types. This folate receptor internalizes folate through caveolae by a process named potocytosis, which is distinct from endocytosis, through clathrin-coated pits. When folate conjugated to poly-L-lysine was used to deliver the E. coli beta-galactosidase gene into tumor cells overexpressing the folate receptor, only low levels of beta-galactosidase activity were detectable. When a replication-defective adenovirus was coincubated with the DNA/folate complexes, 20 to 30% of the cells stained blue with X-gal and a 1000-fold increase of beta-galactosidase activity was observed. Thus, for high efficient DNA delivery and gene expression via the caveolae system, a potosomal disruption agent is needed. Furthermore, folate-mediated DNA delivery is restricted to tumor cells that highly overexpress the folate receptor, which will permit future development of tumor cell-specific delivery of toxic genes for cancer gene therapy.

Adenoviridae↗

Hepatic gene therapy: efficient gene delivery and expression in primary hepatocytes utilizing a conjugated adenovirus-DNA complex.

Receptor-mediated endocytosis is an effective method for gene delivery into target cells. We have previously shown that DNA molecules complexed with asialoglycoprotein can be efficiently endocytosed by primary hepatocytes and the internalized DNA can be released from endosomes by the use of a replication-defective adenovirus. Because the DNA and virus enter target cells independently, activity enhancement requires high concentrations of adenoviral particles. In this study, adenoviral particles were chemically conjugated to poly(L-lysine) and bound ionically to DNA molecules. Quantitative delivery to primary hepatocytes was achieved with significantly reduced viral titer when the asialoorosomucoid-poly(L-lysine) conjugate was included in the complex. The conjugated adenovirus was used to deliver a DNA vector containing canine factor IX to mouse hepatocytes, resulting in the expression of significant concentrations of canine factor IX in the culture medium. The results suggest that receptor-mediated endocytosis coupled with an efficient endosomal lysis vector should permit the application of targeted and efficient gene delivery into the liver for gene therapy of hepatic deficiencies.

Adenoviridae↗

Hepatic gene therapy: adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes.

We have combined a receptor-mediated DNA delivery system with the endosomal lysis ability of adenovirus and shown that DNA can be delivered into primary hepatocytes, resulting in a high level of gene expression. When asialoorosomucoid conjugated with poly(L-lysine) was used to deliver the Escherichia coli beta-galactosidase gene into primary hepatocytes through binding with the hepatic asialoglycoprotein receptor, only a low level of beta-galactosidase was detectable, with less than 0.1% of the hepatocytes being transfected. This level of activity can be greatly enhanced by the cointernalization of the DNA.protein complex with a replication-defective adenovirus, resulting in 100% of the hepatocytes staining blue with 5-bromo-4-chloro-3-indolyl beta-D-galactoside. Quantitative analysis of beta-galactosidase expression also showed a 1000-fold enhancement of activity. To test the applicability of this DNA delivery system for the correction of phenylketonuria, a metabolic disorder that causes severe mental retardation in children, we have delivered the human phenylalanine hydroxylase (PAH) gene to hepatocytes derived from a PAH-deficient mouse strain and demonstrated complete reconstitution of enzymatic activity. This method shows great promise for efficient gene delivery to the liver for correction of hepatic disorders.

3T3 Cells↗