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J Ray

Publications and source records attributed to J Ray.

At least 55 records · Page 3Linked to original sources
PubMed1994

Somatic gene transfer to the adult primate central nervous system: in vitro and in vivo characterization of cells genetically modified to secrete nerve growth factor.

Somatic gene transfer offers a means of delivering substances to the central nervous system (CNS) in a regionally specific, high-dose, chronic and well-tolerated manner. Studies in rats have shown that genetically modified cell grafts can prevent neuronal degeneration and promote functional recovery after injury and can improve cognitive function in aged subjects. To assess the potential utility of somatic gene transfer techniques in primate models, retroviral vectors were used to modify genetically monkey and human primary skin fibroblasts to produce and secrete human nerve growth factor (NGF). In vitro, all cell types produced NGF and sustained this production through cell growth to confluency, as determined by both Northern blot analysis and ELISA. Adult human fibroblasts produced as much NGF as did young human fibroblasts. Monkey fibroblasts genetically modified to produce NGF were then grafted to intact adult rhesus and cynomolgous monkey brains. Among nine primates that received a total of 76 grafts, surviving cells were found in all subjects up to the maximal experimental timepoint of 6 months. Cholinergic fibres from the host brain penetrated NGF-secreting grafts up to 6 months after grafting, but showed little penetration in control grafts lacking the NGF gene. Autografts survived better than allografts. These findings indicate that both human and primate fibroblasts can be transduced to produce and secrete NGF, can maintain this production whether in a growing or quiescent state and can elicit robust sprouting responses when primate fibroblasts are grafted to the adult brain. Cells genetically modified to produce trophic factors are a useful model for studying in vitro and in vivo CNS plasticity and for testing potential therapies for neurodegenerative conditions.

Animals↗
PubMed1994

Grafting of nerve growth factor-producing fibroblasts reduces behavioral deficits in rats with lesions of the nucleus basalis magnocellularis.

Rats received bilateral lesions of the nucleus basalis magnocellularis by infusion of biotenic acid. Two weeks after the lesion, a suspension of genetically modified primary rat fibroblasts was grafted dorsal to the nucleus basalis magnocellularis (2 x 10(5) cells per side). The fibroblasts were either infected with the gene for human beta-nerve growth factor or Escherichia coli beta-galactosidase. The nerve growth factor-producing fibroblasts released 67 ng nerve growth factor/10(5) cells per day in vitro. Two weeks after implantation of the fibroblasts, spatial learning was tested in the Morris water-maze. Nerve growth factor-producing fibroblasts, but not beta-galactosidase-producing fibroblasts ameliorated the deficit in acquisition of the water-maze task. In addition, spatial acuity was improved to near-normal levels by the nerve growth factor-producing grafts. Choline acetyltransferase activity in cortical areas and hippocampus was not affected by the nerve growth factor-producing grafts. Both grafted groups showed a similar reduction in the level of dopamine, but not homovanillic acid or 3-methoxytyramine, in the frontal cortex. Levels of norepinephrine, epinephrine and serotonin and their metabolites in the neocortex and hippocampus were not affected by the lesion or the grafts. Nerve growth factor-producing grafts increased the size of remaining nerve growth factor-receptor (p75) immunoreactive neurons in the nucleus basalis magnocellularis by 25%. Nucleus basalis magnocellularis lesions reduced the integrated optic density of choline acetyltransferase-positive fiber staining in the ventral neocortex by 46%, but nerve growth factor-producing grafts restored this area to 86% of control. These data suggest that nerve growth factor-producing grafts can cause a marked behavioral improvement, probably through the partial restoration of the lesioned projection from nucleus basalis magnocellularis to neocortex.

Animals↗
PubMed1994

In vivo and ex vivo gene transfer to the brain.

The use of gene transfer techniques to express novel proteins within different cellular populations has provided insights into the function and plasticity of the brain. Recently, this technique has been successfully used to explore physiological processes within the CNS and to intervene in neurodegenerative disease and cancer. Progress in manipulating transgene products in vivo and in achieving cell-specific targeting of genetic material offers promise for enhancing the usefulness of this technique and its therapeutic potential for treating human disorders of the CNS.

Animals↗
PubMed1994

Alternative splice site selection in the human growth hormone gene transcript and synthesis of the 20 kDa isoform: role of higher order transcript structure.

Expression of the human growth hormone (hGH) gene in somatotrophs of the anterior pituitary gland results in the synthesis and secretion of a major 22 kDa and a minor 20 kDa GH isohormone. The expression of these two proteins reflects the alternative utilization of a major (B) and a minor (B') splice acceptor site in exon 3 of the hGH-N transcript. By comparing the structure and splicing patterns of the hGH-N gene transcript with that of the structurally related, placentally expressed, hGH-V gene transcript, which uses only the major (B) exon 3 splice acceptor, it has been possible to define the cis-acting elements in exon 3 that are critical for activation of the B' splice acceptor. The present paper demonstrates that, in addition to the importance of sequences in the immediate proximity of the two alternative splice acceptor sites, additional more remote sequences in the transcript also contribute to this alternative splice site selection. The data further suggest that these more distal sequences do not act individually, but interact so that the net level of alternative splicing in exon 3 is dictated by the overall higher order structure of the hGH-N transcript.

Alternative Splicing↗
PubMed1994

Spinal cord neuroblasts proliferate in response to basic fibroblast growth factor.

Trophic factors may function as one of the epigenic signals responsible for the proliferation, growth, migration, and differentiation of neurons and glia during embryogenesis. The present study reports that basic fibroblast growth factor (bFGF) at high concentrations (10-100 ng/ml) is a mitogen for embryonic spinal cord cells that have already committed to a neuronal pathway and are expressing neuronal phenotypes (neuroblasts). Neuroblasts proliferate with a doubling time of 2.5 d. To characterize the nature of cells proliferating in response to bFGF, we have established long-term cultures of neuroblasts that can be passaged, freeze thawed, and recultured. In cultures the proportion of astrocytes remained the same, indicating limited survival and proliferation of these cells in response to bFGF. These results indicate that bFGF has mitogenic effects preferably on neuroblasts. The morphological and biochemical characterizations of the neuronal populations present in the long-term neuroblast cultures are presented here. The presence of cholinergic and GABAergic neurons in the cultures was established by immunocytochemical analysis. The cultures contain a small number of motoneurons as judged by their immunostaining with ChAT, low-affinity NGF receptor (LNGFR), and large size. Among all other growth factors tested for their mitogenic effects on embryonic spinal cord cells in culture, only epidermal growth factor (EGF) showed such effects, but to a lesser degree. The proliferative nature of neuroblasts has made it possible to transduce the Escherichia coli beta-galactosidase (LacZ) gene stably into these cells in vitro using a retroviral vector. The transfected cells expressing the foreign gene can be passaged, freeze thawed, and recultured without the loss of transgenes. The ability to transduce foreign genes stably into these cells permits implantation of these cells in the spinal cord to study cellular and biochemical behaviors and gene expression in defined neuronal populations in in vivo environments.

Animals↗
PubMed1993

Proliferation, differentiation, and long-term culture of primary hippocampal neurons.

Primary embryonic hippocampal neurons can develop morphologically and functionally in culture but do not survive more than a few weeks. It has been reported that basic fibroblast growth factor (bFGF) promotes the survival of and neurite elongation from fetal hippocampal neurons. We report that bFGF, in a dose-dependent manner, can induce the survival (50 pg to 1 ng/ml) and proliferation (10-20 ng/ml) of embryonic hippocampal progenitor neurons in vitro. In serum-free medium containing high concentrations of bFGF, neurons not only proliferated (4-day doubling time) and differentiated morphologically but also could be passaged and grown as continuous cell lines. The neuronal nature of the proliferating cells was positively established by immunostaining with several different neuron-specific markers and by detailed ultrastructural analyses. The proliferative effect of bFGF was used to generate nearly pure neuronal cell cultures that can be passaged, frozen, thawed, and cultured again. Neurons have been maintained > 5 months in culture. The ability to establish long-term primary neuronal cultures offers the possibility that clonal lines of distinct neuronal cell types may be isolated from specific areas of the central nervous system. Such long-term neuronal cultures should prove valuable in studying neurons at the individual cell level and also in exploring interactions between neurons in vitro. The observed dose dependence raises the possibility that cell survival and proliferation in vivo may be influenced by different levels of bFGF.

Animals↗
PubMed1993

Thymidine kinase-mediated killing of rat brain tumors.

Gene therapy has many potential applications in central nervous system (CNS) disorders, including the selective killing of tumor cells in the brain. A rat brain tumor model was used to test the herpes simplex virus (HSV)-thymidine kinase (TK) gene for its ability to selectively kill C6 and 9L tumor cells in the brain following systemic administration of the nucleoside analog ganciclovir. The HSV-TK gene was introduced in vitro into tumor cells (C6-TK and 9L-TK), then these modified tumor cells were evaluated for their sensitivity to cell killing by ganciclovir. In a dose-response assay, both C6-TK and 9L-TK cells were 100 times more sensitive to killing by ganciclovir (median lethal dose: C6-TK, 0.1 microgram ganciclovir/ml; C6, 5.0 micrograms ganciclovir/ml) than unmodified wild-type tumor cells or cultured fibroblasts. In vivo studies confirmed the ability of intraperitoneal ganciclovir administration to kill established brain tumors in rats as quantified by both stereological assessment of brain tumor volumes and studies of animal survival over 90 days. Rats with brain tumors established by intracerebral injection of wild-type or HSV-TK modified tumor cells or by a combination of wild-type and HSV-TK-modified cells were studied with and without ganciclovir treatments. Stereological methods determined that ganciclovir treatment eliminated tumors composed of HSV-TK-modified cells while control tumors grew as expected (p < 0.001). In survival studies, all 10 rats with 9L-TK tumors treated with ganciclovir survived 90 days while all untreated rats died within 25 days. Curiously, tumors composed of combinations of 9L and 9L-TK cells could be eliminated by ganciclovir treatments even when only one-half of the tumor cells carried the HSV-TK gene. While not completely understood, this additional tumor cell killing appears to be both tumor selective and local in nature. It is concluded that HSV-TK gene therapy with ganciclovir treatment does selectively kill tumor cells in the brain and has many potential applications in CNS disorders, including the treatment of cancer.

Animals↗
PubMed1993

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Breech Presentation↗
PubMed1992

Theory of delocalized ionic binding to polynucleotides: structural and excluded-volume effects.

A previously developed theory for the delocalized binding of ions to polyelectrolytes was restricted to point ions and a structurally rigid polyelectrolyte. For the binding of substances like oligolysines and polyamines to DNA, the restriction to point ions would appear not to be realistic. For the binding of ions to flexible chains like single-stranded polynucleotides, the restriction to a rigid polyelectrolyte may not be realistic. In this article, we assess the effect of relaxation of these two restrictions. Excluded volume among bound ions is modeled by a hard-rod potential in the context of the theory of a one-dimensional fluid. The possibility that a flexible chain folds in some manner in the immediate vicinity of a bound ion is modeled by allowing the mean spacing between charged groups on the polymer to become smaller as the number of bound ions increases. We compare our results with recent data on the binding of a series of oligolysines to single-stranded polynucleotides, which conflict with the predictions of the original theory of delocalized binding of point ions to rigid polyelectrolytes. Inclusion of excluded volume among bound ions does not significantly improve agreement with the data. Substantial improvement in the level of agreement is obtained when the polyion chain is assumed to be flexible. One of our conclusions is that the excluded-site description of anticooperativity, which was designed for the binding of ligands to discrete sites on a polymer chain, and which does not include the effect of ionic forces, should not be used in cases of delocalized binding of ions.

Binding Sites↗
PubMed1992

Coronary venous intimal thickening in explanted cardiac allografts. Evidence demonstrating that transplant coronary artery disease is a manifestation of a diffuse allograft vasculopathy.

The accelerated coronary artery disease occurring in cardiac allografts is thought to be a form of chronic rejection directed against allogeneic vascular endothelium. If this hypothesis is correct, one would anticipate disease not only in the arteries but in venous structures as well. Accordingly, the degree of myointimal proliferation of both coronary arteries and coronary veins was assessed in 22 explanted or autopsied cardiac allografts by light microscopy. Other factors assessed included clinical cause of death/retransplantation, time posttransplantation, underlying cardiac disease, donor and recipient age, and ischemic time. Thirteen of the 22 hearts had either moderate or severe arterial myointimal thickening. Of these, 10 hearts had associated coronary venous thickening. Of the 9 remaining hearts with either mild or no arterial myointimal thickening, none had venous involvement. The overall correlation between the presence and degree of allograft coronary artery and allograft coronary vein thickening was high (r = 0.80, P = 0.0014). Of the other demographic factors investigated, only length of time posttransplant had a weak correlation with arterial or venous myointimal thickening (r = 0.46, P = 0.045 and r = 0.48, P = 0.039, respectively. These data demonstrate that the usually termed "accelerated transplant atherosclerosis" in the cardiac allograft is a true vasculopathy and involves both the arterial and venous systems.

Adult↗
PubMed1992

Gene transfer into established and primary fibroblast cell lines: comparison of transfection methods and promoters.

The stable transfection of immortalized Rat-1 and rat skin primary fibroblast cell lines by calcium phosphate precipitation, lipofection and electroporation methods have been examined. The lipofection method was found to be better than the other methods in terms of higher transfection efficiency and convenient use. Expression of beta-galactosidase from two different viral promoters showed that the level of transgene expression depends on the promoter strength in a particular cell type. The results presented here show that the transgene expression is extremely variable among different colonies generated from individually transfected cells. Therefore, it is necessary to examine individual colonies of cells for the production of reporter gene to obtain cell lines expressing high amounts of gene products.

Animals↗
PubMed1991

Cellular replacement therapy for neurologic disorders: potential of genetically engineered cells.

Neural transplantation, a mode of cellular replacement, has been used as a therapeutic trial for Parkinson's disease. Studies indicate that tonic release of the metabolites from the graft that can be utilized by the host brain, is likely to be the major mechanism responsible for the therapeutic effect. The use of fetal tissue is complicated by ethical controversy and immunological incompatibility. Autografting adult tissue has not been successful mainly due to poor survival. Genetically engineered cells are promising alternative sources of donor cells. We have investigated the potential of primary skin fibroblasts as donor cells for intracerebral grafting. Primary skin fibroblasts survive in the brain and remain in situ. A number of genes (nerve growth factor, tyrosine hydroxylase, glutamic acid decarboxylase, and choline acetyltransferase) have been successfully introduced and expressed in the primary fibroblasts. The L-dopa-secreting primary fibroblasts exhibited a behavioral effect in a rat model of Parkinson's disease up to 8 weeks after being grafted into denervated striatum. Factors that can maximize gene transfer, transgene expression, and fibroblast survival in the brain make up the future direction of investigation.

Animals↗
PubMed1991

Employment of fibroblasts for gene transfer: applications for grafting into the central nervous system.

Genetic modification of primary skin fibroblasts offers a new approach to the focal delivery of deficient transmitter-specific enzymes (e.g., TH) or trophic substances (e.g., NGF) to the damaged or diseased CNS. Although fibroblasts are unable to provide anatomical corrections to defective neural connectivity, they can serve as biological pumps for the enzymes and growth factors in vivo. The capability of genetically engineered cells to ameliorate disease phenotypes in animal models of CNS disorders may ultimately results in the restoration of function. At this time, primary skin fibroblasts appear to be a convenient cellular population for the application of gene transfer and intracerebral grafting for the animal model of Parkinson's disease. It is now important for future investigations to provide data concerning the long-term stable expression of the transgene product (e.g., TH) following intracerebral implantation, as well as determining optimal conditions for the survival of primary cells grafted into the nervous system.

Animals↗
PubMed1991

Rapid accumulation of measles virus leader RNA in the nucleus of infected HeLa cells and human lymphoid cells.

The 3' terminus of the single-stranded, negative-sense genome of the measles virus comprises a 55-nucleotide-long sequence, which is transcribed into a short, positive-sense RNA called the leader sequence. In other viral systems, this RNA has been shown to modulate host cell transcription. Here, we report the presence of measles virus leader RNA in both cytoplasmic and nuclear fractions of infected HeLa cells as well as T- and B-lymphoid cells. A sharp and rapid increase in the concentration of leader RNA in the nucleus of infected HeLa cells was also observed. The presence and accumulation of leader RNA in the nucleus of infected cells supports the hypothesis that the leader RNA plays a role in the down regulation of host cell transcription and may be responsible for the suppression of immunoglobulin synthesis by measles virus-infected B cells. Such alterations in immune responsiveness could aid in the establishment of a persistent infection by measles virus.

Base Sequence↗
PubMed1991

Human growth hormone-variant is a biologically active somatogen and lactogen.

The human GH-variant (hGH-V) gene, a member of the GH-PRL gene family, is expressed by the placenta during the second and third trimesters of gestation. The secreted hGH-V protein differs from pituitary GH (hGH-N) by only 13 amino acids. We have previously demonstrated that hGH-V can bind to both somatogen and lactogen cell surface receptors in vitro, but that the ratio of its somatogen to lactogen receptor-binding affinities is substantially higher than that of hGH-N. We now characterize the somatogen and lactogen bioactivities of hGH-V and contrast them to the bioactivity of hGH-N. Somatogen bioactivity was assayed by stimulation of weight gain in hypophysectomized rats, and lactogen bioactivity was assayed by the mitogenic response of the Nb2 lymphoma cell line. While the average increase in rat body weight in response to a fixed concentration of hormone was comparable using either hGH-V or hGH-N, the mitotic response of the lactogen-inducible Nb2 cells was significantly less for hGH-V. The comparable somatogen, but lower lactogen, bioactivity of hGH-V relative to hGH-N parallels the previously reported receptor binding profiles of the two hormones and suggests that hGH-V has the potential to perform a unique role during human gestation.

Animals↗
PubMed1991

Human growth hormone variant produces insulin-like and lipolytic responses in rat adipose tissue.

Genes for normal human pituitary GH (hGH-N) and the GH variant (hGH-V) were expressed in stably transfected mouse mammary cells. The biological properties of hGH-N and hGH-V secreted into the medium were examined using rat adipocytes or epididymal fat segments. Methionyl-hGH produced in E. coli served as a reference standard. The three preparations were quite similar in their ability to bind specifically to intact fat cells and were virtually indistinguishable in their ability to increase glucose oxidation (an insulin-like response), induce refractoriness to insulin-like stimulation, and induce lipolysis in the presence of glucocorticoid. We conclude that placentally expressed hGH-V has a spectrum of metabolic activity comparable to pituitary hGH-N and may contribute to regulation of carbohydrate and lipid metabolism during pregnancy.

Adipose Tissue↗