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J R Morgan

Publications and source records attributed to J R Morgan.

At least 19 recordsLinked to original sources
PubMed2006

Clathrin and synaptic vesicle endocytosis: studies at the squid giant synapse.

The role of clathrin-mediated endocytosis in SV (synaptic vesicle) recycling has been studied by combining molecular biology, physiology and electron microscopy at the squid giant synapse. Procedures that prevent clathrin from assembling into membrane coats, such as impairment of binding of the AP180 and AP-2 adaptor proteins, completely prevent membrane budding during endocytosis. These procedures also reduce exocytosis, presumably an indirect effect of a reduction in the number of SVs following block of endocytosis. Disrupting the binding of auxilin to Hsc70 (heat-shock cognate 70) prevents clathrin-coated vesicles from uncoating and also disrupts SV recycling. Taken together, these results indicate that a clathrin-dependent pathway is the primary means of SV recycling at this synapse under physiological conditions.

Animals↗
PubMed2005

Origin of insulin secreted from islet-like cell clusters derived from murine embryonic stem cells.

Islet-like cell clusters (ILCCs) were derived from murine embryonic stem cells using a slightly modified version of the protocol originally described by Lumelsky et al. in 2001. Analysis with enzyme-linked immunosorbent assays (ELISAs) that distinguish human from murine insulin demonstrated that insulin released from these ILCCs, upon initial in vitro glucose challenge, was of non-murine origin and in fact corresponded to the species of insulin, human or bovine, that had been added to the culture media used to derive ILCCs. This finding convincingly supports the hypothesis that ILCCs are not synthesizing insulin de novo, but rather simply regurgitating insulin taken up during tissue culture. In further experiments, ILCCs were derived in media in which insulin had been replaced by IGF-I with which it shares a common signaling pathway. These ILCCs failed to release any detectable insulin. In contrast, ILCCs produced by various protocols stained positive (dithizone and immunoselective antibodies) for intracellular insulin and, in some cases, C-peptide. Despite the presence of at least some level of de novo, synthesized insulin in ILCCs, the majority of insulin released by ILCCs was sequestered from the exogenous medium.

Animals↗
PubMed2001

Uncoating of clathrin-coated vesicles in presynaptic terminals: roles for Hsc70 and auxilin.

We have examined the roles of Hsc70 and auxilin in the uncoating of clathrin-coated vesicles (CCVs) during neuronal endocytosis. We identified two peptides that inhibit the ability of Hsc70 and auxilin to uncoat CCVs in vitro. When injected into nerve terminals, these peptides inhibited both synaptic transmission and CCV uncoating. Mutation of a conserved HPD motif within the J domain of auxilin prevented binding to Hsc70 in vitro and injecting this mutant protein inhibited CCV uncoating in vivo, demonstrating that the interaction of auxilin with Hsc70 is critical for CCV uncoating. These studies establish that auxilin and Hsc70 participate in synaptic vesicle recycling in neurons and that an interaction between these proteins is required for CCV uncoating.

Adaptor Proteins, Vesicular Transport↗
PubMed2001

Complexation of retrovirus with cationic and anionic polymers increases the efficiency of gene transfer.

Previously, we have demonstrated that chondroitin sulfate proteoglycans and glycosaminoglycans inhibit retrovirus transduction. While studying the mechanism of inhibition, we found that the combined addition of equal-weight concentrations (80 microg/ml) of Polybrene and chondroitin sulfate C to retrovirus stocks resulted in the formation of a high-molecular-weight retrovirus-polymer complex that could be pelleted by low-speed centrifugation. The pelleted complex contained more than 80% of the virus particles, but less than 0.3% of the proteins that were originally present in the virus stock. Surprisingly, the virus in the complex remained active and could be used to transduce cells. The titer of the pelleted virus, when resuspended in cell culture medium to the starting volume, was three-fold greater than the original virus stock. The selectivity (CFU/mg protein) of the process with respect to virus activity was more than 1000-fold. When the pelleted virus-polymer complex was resuspended in one-eighth of the original volume and used to transduce NIH 3T3 murine fibroblasts and primary human fibroblasts, gene transfer was increased 10- to 20-fold over the original unconcentrated retrovirus stock. The implications of our findings for the production, processing, and use of retrovirus stocks for human gene therapy protocols are discussed.

3T3 Cells↗
PubMed2001

Initial experience with a composite autologous skin substitute.

Patients with large burns are surviving in increasing numbers, but there remains no durable and reliable permanent skin replacement. After initial favorable small animal experiments, a pilot trial of a composite skin replacement was performed in patients with massive burns. A composite skin replacement (CSR) was developed by culturing autologous keratinocytes on acellular allogenic dermis. This material was engrafted in patients with massive burns and compared to a matched wound covered with split thickness autograft. With human studies committee approval, 12 wounds in 7 patients were grafted with CSR while a matched control wound was covered with split thickness autograft. These 7 children had an average age of 6.4+/-1.4 yr and burn size of 75.9+/-5.0% of the body surface. Nine wounds were acute burns and three were reconstructive releases. Successful vascularization at 14 days averaged 45.7+/-14.2% (range 0-100%) in the study wounds and 98+/-1% (range 90-100%) in the control sites (P<0.05). Reduced CSR take seemed to correlate with wound colonization. All children survived. While CSR did not engraft with the reliability of standard autograft, this pilot experience is encouraging in that successful wound closure with this material is possible, if not yet dependable. It is hoped that a more mature epidermal layer may facilitate engraftment, and trials to explore this possibility are in progress.

Burns↗
PubMed2001

Keratinocyte growth factor induces hyperproliferation and delays differentiation in a skin equivalent model system.

Keratinocyte growth factor (KGF) is a paracrine mediator of epithelial cell growth. To examine the direct effects of KGF on the morphogenesis of the epidermis, we generated skin equivalents in vitro by seeding human keratinocytes on the papillary surface of acellular dermis and raising them up to the air-liquid interface. KGF was either added exogenously or expressed by keratinocytes via a recombinant retrovirus encoding KGF. KGF induced dramatic changes to the 3-dimensional organization of the epidermis including pronounced hyperthickening, crowding, and elongation of the basal cells, flattening of the rete ridges, and a ripple-like pattern in the junction of stratum corneum and granular layers. Quantitative immunostaining for the proliferation antigen, Ki67, revealed that in addition to increasing basal proliferation, KGF extended the proliferative compartment by inducing suprabasal cell proliferation. KGF also induced expression of the integrin alpha 5 beta 1 and delayed expression of keratin 10 and transglutaminase. However, barrier formation of the epidermis was not disrupted. These results demonstrate for the first time that a single growth factor can alter the 3-dimensional organization and proliferative function of an in vitro epidermis. In addition to new strategies for tissue engineering, such a well-defined system will be useful for analyzing growth factor effects on the complex links between cell proliferation, cell movement and differentiation within a stratified tissue.

Cell Differentiation↗
PubMed2001

Hepatocyte growth factor and melanoma: gene transfer studies in human melanocytes.

Hepatocyte growth factor (HGF), a fibroblast-derived protein that affects the growth, motility and differentiation of epithelial cells, is a mitogen for human melanocytes and has recently been implicated as an important factor for the development and dissemination of melanomas. To better define the possible role of HGF in the multi-step progression from melanocyte to melanoma cell, we used retrovirus-mediated gene transfer to introduce the gene encoding human HGF into normal human melanocytes, thus causing these cells to produce a growth factor they do not normally express and creating a new autocrine loop as sometimes occurs in melanoma cells. Cells were transduced with an efficiency of 6%, and the modified cells synthesized and secreted significant levels of HGF (6.6 ng/10(7) cells per 24 h) in vitro. Cells expressing HGF had a higher rate of proliferation when compared with unmodified cells and formed large, dense, melanin-positive colonies on a plastic surface. Immunostaining showed HGF-positive melanocytes with varying levels of expression, and HGF protein was detected throughout the whole cell. Although proliferation of HGF-expressing melanocytes was enhanced, they failed to form colonies in a soft agar assay. These results suggest that expression of HGF, by virtue of its ability to enhance proliferation and cell clustering, may play a role in the multi-step process of transformation, but an autocrine signal of HGF alone is not sufficient for malignant transformation.

Cell Differentiation↗
PubMed2000

A conserved clathrin assembly motif essential for synaptic vesicle endocytosis.

Although clathrin assembly by adaptor proteins (APs) plays a major role in the recycling of synaptic vesicles, the molecular mechanism that allows APs to assemble clathrin is poorly understood. Here we demonstrate that AP180, like AP-2 and AP-3, binds to the N-terminal domain of clathrin. Sequence analysis reveals a motif, containing the sequence DLL, that exists in multiple copies in many clathrin APs. Progressive deletion of these motifs caused a gradual reduction in the ability of AP180 to assemble clathrin in vitro. Peptides from AP180 or AP-2 containing this motif also competitively inhibited clathrin assembly by either protein. Microinjection of these peptides into squid giant presynaptic terminals reversibly blocked synaptic transmission and inhibited synaptic vesicle endocytosis by preventing coated pit formation at the plasma membrane. These results indicate that the DLL motif confers clathrin assembly properties to AP180 and AP-2 and, perhaps, to other APs. We propose that APs promote clathrin assembly by cross-linking clathrin triskelia via multivalent interactions between repeated DLL motifs in the APs and complementary binding sites on the N-terminal domain of clathrin. These results reveal the structural basis for clathrin assembly and provide novel insights into the molecular mechanism of clathrin-mediated synaptic vesicle endocytosis.

Adaptor Protein Complex alpha Subunits↗
PubMed2000

[Gene therapy possibilities in plastic surgery].

Advances in gene technologies have meanwhile reached plastic surgery. Important contributions in this field (which are not all included in the paper) come not only from plastic surgeons, but also from neighboring specialities like dermatology, trauma surgery, orthopedics and vascular surgery. The uniting principle for all this work is improving wound healing and reconstructing tissue defects taking into consideration functional and aesthetic aspects. Gene-therapy is gaining further importance in the clinical field of plastic surgery. In this regard, every clinician has to be aware of the fact that progress in experimental and experimental-clinical work will be achieved only with the help of basic science. On the other hand, basic science needs the clinical input to get relevant patient-oriented studies started. Further intensive cooperation between clinicians and basic scientists is therefore mandatory. In plastic surgery, 2 years ago we founded a forum called ECSAPS (European Conference of Scientists and Plastic Surgeons), which takes place in European city every year.

Animals↗
PubMed2000

Plasmin triggers rapid contraction and degradation of fibroblast-populated collagen lattices.

We examined the role of the serine proteinase plasmin in regulating fibroblast-mediated tissue remodeling during wound healing. As an in vitro model system, collagen lattices were seeded with human dermal fibroblasts, and various concentrations of plasmin were added to the medium of the contracting lattices. Within 16 h, fibroblast-populated collagen lattices treated with plasmin rapidly contracted from approximately 20 mm to less than 2 mm in diameter. Measurements of collagen lattices with radiolabeled collagen indicated that, when these lattices included either fibroblasts or conditioned medium derived from fibroblast-populated collagen lattices, exogenous plasmin induced collagen degradation and rapid lattice contraction. Western blot analyses of conditioned medium demonstrated that fibroblasts in collagen lattices secreted the latent matrix metalloproteinase, MMP-1, which was subsequently cleaved by plasmin. Additionally, rapidlattice contraction and collagen degradation were blocked when collagen lattices were treated simultaneously with plasmin and aprotinin or a tissue inhibitor of metalloproteinases, TIMP-1. These results provide strong evidence that plasmin regulates rapid contraction of collagen lattices by activating fibroblast-secreted MMP-1 that triggers collagen degradation. The findings from this study suggest that fibroblast-populated collagen lattices can be used as an in vitro model system to investigate the mechanisms by which plasmin and cell-secreted plasminogen activators control MMP-1 mediated extracellular lattice degradation and remodeling during wound healing.

Aprotinin↗
PubMed2000

Microfabrication of an analog of the basal lamina: biocompatible membranes with complex topographies.

A microfabrication approach was used to produce novel analogs of the basal lamina with complex topographic features. A test pattern of ridges and channels with length scales (40 to 310 micrometer) similar to the invaginations found in a native basal lamina was laser machined into the surface of a polyimide master chip. Negative replicates of the chip were produced using polydimethylsiloxane silicone elastomer and these replicates were used as templates for the production of thin ( approximately 21 micrometer) membranes of collagen or gelatin. The resulting membranes had a complex topography of ridges and channels that recapitulated the features of the master chip. To demonstrate their utility, these complex membranes were laminated to type I collagen sponges and their surfaces were seeded with cultured human epidermal keratinocytes to form a skin equivalent. The keratinocytes formed a differentiated and stratified epidermis that conformed to the features of the microfabricated membrane. The topography of the membrane influenced the differentiation of the keratinocytes because stratification was enhanced in the deeper channels. Membrane topography also controlled the gross surface features of the skin equivalent; infolds of the epidermis increased as channel depth increased. These novel microfabricated analogs of the basal lamina will help to elucidate the influence of topography on epithelial cell proliferation and differentiation and should have applications in the tissue engineering of skin equivalents as well as other basal lamina-containing tissues.

Basement Membrane↗
PubMed2000

Toward a more accurate quantitation of the activity of recombinant retroviruses: alternatives to titer and multiplicity of infection.

In this paper, we present a mathematical model with experimental support of how several key parameters govern the adsorption of active retrovirus particles onto the surface of adherent cells. These parameters, including time of adsorption, volume of virus, and the number, size, and type of target cells, as well as the intrinsic properties of the virus, diffusion coefficient, and half-life (t(1/2)), have been incorporated into a mathematical expression that describes the rate at which active virus particles adsorb to the cell surface. From this expression, we have obtained estimates of C(vo), the starting concentration of active retrovirus particles. In contrast to titer, C(vo) is independent of the specific conditions of the assay. The relatively slow diffusion (D = 2 x 10(-8) cm(2)/s) and rapid decay (t(1/2) = 6 to 7 h) of retrovirus particles explain why C(vo) values are significantly higher than titer values. Values of C(vo) also indicate that the number of defective particles in a retrovirus stock is much lower than previously thought, which has implications especially for the use of retroviruses for in vivo gene therapy. With this expression, we have also computed AVC (active viruses/cell), the number of active retrovirus particles that would adsorb per cell during a given adsorption time. In contrast to multiplicity of infection, which is based on titer and is subject to the same inaccuracies, AVC is based on the physicochemical parameters of the transduction assay and so is a more reliable alternative.

3T3 Cells↗
PubMed2000

Toward a more accurate quantitation of the activity of recombinant retroviruses: alternatives to titer and multiplicity of infection.

In this paper, we present a mathematical model with experimental support of how several key parameters govern the adsorption of active retrovirus particles onto the surface of adherent cells. These parameters, including time of adsorption, volume of virus, and the number, size, and type of target cells, as well as the intrinsic properties of the virus, diffusion coefficient, and half-life (t1/2), have been incorporated into a mathematical expression that describes the rate at which active virus particles adsorb to the cell surface. From this expression, we have obtained estimates of Cvo, the starting concentration of active retrovirus particles. In contrast to titer, Cvo is independent of the specific conditions of the assay. The relatively slow diffusion (D = 2 x 10(-8) cm2/s) and rapid decay (t1/2 = 6 to 7 h) of retrovirus particles explain why Cvo values are significantly higher than titer values. Values of Cvo also indicate that the number of defective particles in a retrovirus stock is much lower than previously thought, which has implications especially for the use of retroviruses for in vivo gene therapy. With this expression, we have also computed AVC (active viruses/cell), the number of active retrovirus particles that would adsorb per cell during a given adsorption time. In contrast to multiplicity of infection, which is based on titer and is subject to the same inaccuracies, AVC is based on the physicochemical parameters of the transduction assay and so is a more reliable alternative.

Animals↗
PubMed1999

A role for the clathrin assembly domain of AP180 in synaptic vesicle endocytosis.

We have used the squid giant synapse to determine whether clathrin assembly by AP180 is important for synaptic vesicle endocytosis. The squid homolog of AP180 encodes a 751 amino acid protein with 40% sequence identity to mouse AP180. Alignment of squid AP180 with other AP180 homologs shows that amino acid identity was highest in the N-terminal inositide-binding domain of the protein and weakest in the C-terminal clathrin assembly domain. Recombinant squid AP180 was able to assemble clathrin in vitro, suggesting a conserved three-dimensional structure that mediates clathrin assembly despite the divergent primary sequence of the C-terminal domain. Microinjection of the C-terminal domains of either mouse or squid AP180 into the giant presynaptic terminal of squid enhanced synaptic transmission. Conversely, a peptide from the C-terminal domain of squid AP180 that inhibited clathrin assembly in vitro completely blocked synaptic transmission when it was injected into the giant presynaptic terminal. This inhibitory effect occurred over a time scale of minutes when the synapse was stimulated at low (0.03 Hz), physiological rates. Electron microscopic analysis revealed several structural changes consistent with the inhibition of synaptic vesicle endocytosis; peptide-injected terminals had far fewer synaptic vesicles, were depleted of coated vesicles, and had a larger plasma membrane perimeter than terminals injected with control solutions. In addition, the remaining synaptic vesicles were significantly larger in diameter. We conclude that the clathrin assembly domain of AP180 is important for synaptic vesicle recycling at physiological rates of activity and that assembly of clathrin by AP180 is necessary for maintaining a pool of releasable synaptic vesicles.

Adaptor Proteins, Vesicular Transport↗
PubMed1999

Proteins involved in synaptic vesicle trafficking.

Neurotransmitter release relies on a series of synaptic vesicle trafficking reactions. We have determined the molecular basis of these reactions by microinjecting, into 'giant' nerve terminals of squid, probes that interfere with presynaptic proteins. These probes affect neurotransmitter release and disrupt nerve terminal structure. From the nature of these lesions, it is possible to deduce the roles of individual proteins in specific vesicle trafficking reactions. This approach has revealed the function of more than a dozen presynaptic proteins and we hypothesize that neurotransmitter release requires the coordinated action of perhaps 50-100 proteins.

Animals↗
PubMed1999

Kinetics of retrovirus production and decay.

There has been only limited success in using recombinant retroviruses to transfer genes for the purposes of human gene therapy, in part because the average number of genes delivered to the target cells (transduction efficiency) is often too low to achieve the desired therapeutic effect [Miller, AD. 1990. Blood 76:271-278; Mulligan RC. 1993. Science 260:926-932; Orkin SH, Motulsky AG. 1995. Report and recommendations of the panel to assess the NIH investment in research on gene therapy. Bethesda, MD: National Institutes of Health.]. One strategy to improve transduction efficiency is to focus on understanding and improving the processes used to produce recombinant retroviruses. In this report, we characterized the dynamics of retrovirus production and decay in batch cultures of virus producer cells using a simple mathematical model, a recombinant retrovirus encoding the Escherichia coli lacZ gene, and quantitative assays for virus activity and number. We found that the rate at which recombinant retroviruses spontaneously lose their activity (decay) is a strong function of temperature, decreasing roughly 2-fold for every 5 degrees C reduction in temperature, whereas the rate at which retroviruses are produced is only weakly affected by temperature, decreasing about 10% for every 5 degrees C reduction in temperature. In addition, we developed a simple mathematical model of virus production and decay that predicted that the virus titer in batch cultures of virus producer cells would reach a maximum steady-state at a rate that is inversely proportional to the virus decay rate and to a level that is proportional to the ratio of the virus production rate to the virus decay rate. Consistent with the model, we observed that the steady-state levels of virus titer increased more than 3-fold when the cell culture temperature was reduced from 37 to 28 degrees C. Despite their higher titers, virus stocks produced at 28 degrees C, when used in undiluted form so as to mimic human gene transfer protocols, did not transduce substantially more cells than virus stocks produced at 37 degrees C. The implications of our findings on the production of retroviruses for use in human gene therapy protocols are discussed.

3T3 Cells↗
PubMed1999

Uptake of precursor and synthesis of transmitter in a histaminergic photoreceptor.

As a first step in understanding how the supply of the neurotransmitter histamine is maintained in a photoreceptor, we followed the uptake and metabolism of the immediate precursor of histamine, histidine. [3H]Histidine taken up into photoreceptors and glia was detected using autoradiography, and synthesis of [3H]histamine from [3H]histidine was assayed with thin-layer chromatography. Photoreceptors from barnacles were pulsed (15 min) with [3H]histidine (0.2-200 microM), then maintained in normal saline for up to 24 hr. Autoradiography showed that photoreceptor somata, axons, and presynaptic arbors were labeled, but only weakly, like (nonhistaminergic) ganglion cells. Label instead was concentrated over surrounding glia. Stimulating preparations with light did not increase photoreceptor labeling. Grain counts from photoreceptor axons showed uptake of [3H]histidine into these neurons by a Na+-dependent mechanism with a Km of approximately 50 microM. Over 24 hr only 1% of the [3H]histidine taken up by preparations was converted to [3H]histamine either in the dark or in the light. Injections of [3H]histidine directly into photoreceptors established that synthesis takes place within the photoreceptors and confirmed that stimulation with light did not measurably affect the rate of conversion of [3H]histidine to [3H]histamine. These results suggest that de novo synthesis of transmitter is unlikely to be as important as its reuptake in maintaining neurotransmitter supply in these photoreceptor terminals. In support of this conclusion, photoreceptors accumulated more label when transmitter release was stimulated with high K+ and histamine uptake was antagonized with chlorpromazine.

Animals↗