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Biomedical subjects

D M Lam

Publications and source records attributed to D M Lam.

At least 19 recordsLinked to original sources
PubMed1998

Mechanistic analyses of ion dependences in a high-affinity human serotonin transport system in transfected murine fibroblast cells.

1. A clonal cell line, L-S1, has been identified from transfection of human genomic DNA into cultured mouse L-M fibroblasts. Because this transfectant cell line stably expresses a high-affinity serotonin (5-HT) transport mechanism with kinetic and pharmacological properties comparable to those of other serotonin uptake systems, it was used to investigate the mechanistic involvement of Na+ and Cl- ions in the ligand binding and kinetic uptake processes of this system. 2. Intact transfectant cells, when incubated at low temperature (4 C), enabled quantitative assessment of imipramine-displaceable 5-[3H]HT binding to the 5-HT transport system. This binding activity is insensitive to the presence of various ligands specific for 5-HT receptor subtypes. 3. Imipramine-displaceable 5-[3H]HT binding to intact L-S1 cells was shown to be a Cl--dependent but Na+-independent process. Chloride ions lack binding co-operativity in facilitating ligand binding. Changes in external Cl- concentration altered the Kd but not the Bmax of binding. 4. The overall transport activity was observed to be highly dependent on both external Na+ and Cl- concentrations, characterized by a 5-HT:Na+:Cl- coupling ratio of 1:1:1 per transport cycle. Alterations in the external concentrations of both Na+ and Cl- ions altered only the Km and not the Vmax of transport. 5. Both binding and kinetic results are consistent with kinetic modelling predictions of the Cl- ion in facilitating 5-HT binding to the transport system, and of the Na+ ion in enabling translocation of bound 5-HT across the plasma membrane. Thus, Na+ and Cl- ions facilitate mechanistically distinct and discernible functions in the transport cycle.

Animals↗
PubMed1997

Inhibition of cell proliferation on lens capsules by 4197X-ricin A immunoconjugate.

PURPOSE: To evaluate the cytotoxicity of immunotoxin 4197X-ricin A (4197X-RA) and its ability to inhibit protein synthesis and human lens epithelial cell (LEC) proliferation on the inner surface of the lens capsule. SETTING: Houston Biotechnology, Inc., The Woodlands, Texas. METHODS: A cell culture system was established using human LECs as a model for the proliferation of remnant LECs that occurs during posterior capsule opacification (PCO) after extracapsular cataract extraction. The LEC culture system was also used in vitro for testing compounds that might inhibit this process in vivo. Human LECs were cultured on the surface of the original lens capsule fixed to collagen. Variability was reduced by dissecting each lens capsule into equivalent halves and exposing the segments to immunotoxin 4197X-RA. RESULTS: Protein synthesis and LEC proliferation were almost completely inhibited at relatively low 4197X-RA concentrations after short exposure. The inhibitory effects persisted up to 3 weeks after withdrawal of the immunotoxin and after several media exchanges. CONCLUSION: Immunotoxin 4197X-RA may help prevent PCO after primary cataract surgery.

Antibodies, Monoclonal↗
PubMed1993

Molecular cloning and structure of the human (GABATHG) GABA transporter gene.

A cDNA molecule encoding the human GABA transporter was synthesized by means of polymerase chain reaction (PCR) technique and used as probe for selecting a human genomic DNA fragment encoding GABA transporter. A positive clone harboring the whole gene was obtained from a human lymphocyte genomic library through utilizing the genomic 'walking' technique. The clone, designated as pHGAT, harbours a DNA fragment of about 39 kb in length inserted into the BamHI site in cosmid pWE15. The gene covers about 25 kb in length and is constituted by four EcoRI restricted fragments which are 13.7 kb, 3.1 kb, 4.2 kb and 7.2 kb long, respectively. The genomic clone contains 15 introns, including two introns prior to the initiator methionine (i.e., the translation start site is in exon 3). Eleven exons encode the twelve transmembrane regions in the transporter protein. Thus as in the case for a number of other membrane proteins, there appears to be a strong tendency for the putative transmembrane domains to be encoded by separate exons. It is noted that the structure of the human GABA transporter gene reported here differs from the mouse gene which is contains 12 introns.

Base Sequence↗
PubMed1992

Expression of hepatitis B surface antigen in transgenic plants.

Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBsAg was purified from transgenic plants by immunoaffinity chromatography and examined by electron microscopy. Spherical particles with an average diameter of 22 nm were observed in negatively stained preparations. Sedimentation of transgenic plant extracts in sucrose and cesium chloride density gradients showed that the recombinant HBsAg and human serum-derived HBsAg had similar physical properties. Because the HBsAg produced in transgenic plants is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast, which are used as vaccines, we conclude that transgenic plants hold promise as low-cost vaccine production systems.

Cloning, Molecular↗
PubMed1992

Differential regulation of the imipramine-sensitive serotonin transporter by cAMP in human JAr choriocarcinoma cells, rat PC12 pheochromocytoma cells, and C33-14-B1 transgenic mouse fibroblast cells.

The imipramine-sensitive serotonin transporter appears to be the receptor for clinically important antidepressants. Some studies suggest that this protein may fall under the influence of abnormal and as yet uncharacterized regulatory effects during depressive illness. Despite these putative disease-related effects, regulation has never been demonstrated in either the platelet or synaptosome model systems. Here we demonstrate for the first time that the imipramine-sensitive serotonin transport activity in either JAr choriocarcinoma or PC12 pheochromocytoma cell lines is subject to regulation by cAMP. Unexpectedly, the regulatory effect is opposite in the two cases, causing stimulation in JAr (increased Vmax) and inhibition in PC12 (kinetically complex). Appearance of these kinetic effects lagged 15 to 20 hours behind peak cAMP levels. The results are consistent with the interesting possibility that different tissues may express isoforms of the recently cloned serotonin transporter cDNAs. We suggest, therefore, that JAr and PC12 are attractive models in which to pursue detailed analysis of serotonin transport and its manner of regulation by cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Ocular pharmacokinetics of lens epithelial cell-specific immunotoxin 4197X-RA.

The ocular clearance, stability and systemic distribution of a lens epithelial cell-specific immunotoxin were evaluated in adult Dutch Belted rabbits. Immunotoxin 4197X-RA was prepared from a murine monoclonal antibody conjugated by a disulfide linkage to ricin A. After intracameral (i.c.) injection, clearance of the immunotoxin from the eye followed a first-order process. The half-life (t1/2) of immunotoxin in the aqueous humor was 51 min. Accumulation of the immunotoxin in ocular tissues appeared to be associated with aqueous outflow tracts, as it was primarily confined to the iris and the limbal regions of the cornea. No acute metabolism of immunotoxin was observed in the anterior chamber. The immunotoxin was detected in the blood 20 min post-injection and achieved a steady-state level after 40 to 180 min. A slow decline in labeled protein was then observed from 180 min to 24 hr. Systemic clearance of the immunotoxin appeared to occur primarily by way of the kidneys. The present data indicate that immunotoxin 4197X-RA, like other large proteins, is passively cleared from the anterior chamber by aqueous flow, and that little or no acute breakdown occurs in the anterior chamber. It is anticipated that these data, along with results from cytotoxicity studies, will result in the determination of optimal doses and frequency of administration for the use of immunotoxin in human clinical trials.

Animals↗
PubMed1992

Organization and development of horizontal cells in the goldfish retina, II: Use of monoclonal antibody MH1.

We have produced and characterized a monoclonal antibody, MH1, which selectively labels rod horizontal cells and Müller cells in the goldfish retina. Biochemical and tissue distribution studies indicate that MH1 may recognize four out of five classes of intermediate filament proteins in goldfish: vimentin, desmin, glial fibrillary acidic protein (GFAP), and keratin, but not neurofilament. The intermediate filament which is labeled strongest in the retina is vimentin. In the goldfish retina, the only type of horizontal cells recognized by MH1 appear to be rod horizontal cells. This result suggests that the rod horizontal cell, an interneuron, and Müller (glial) cells share a common antigen: vimentin, which is usually only expressed in mesenchymal origin cells. The development of rod horizontal cells in the goldfish retina was also studied using MH1. The cells were not labeled by MH1 until 4-6 weeks posthatching, a stage in which the animals are already visually active. MH1 also did not label any horizontal cell in the region close to the ora terminalis in the goldfish retina. These results suggest that either the emergence and maturation of rod horizontal cells occur late during goldfish retinal development or the expression of vimentin itself occurs late in the development of rod horizontal cells.

Animals↗
PubMed1992

Kelly's hospital ship.

The JN-4 "Jennie" and DH-4 air ambulance conversions used by the U.S. Army during the period 1918-1924 are well-known to those interested in the history of air evacuation. However, another "unknown" craft was also used during this period. The author unravels the mystery of this craft converted for use as an air ambulance at Kelly Field, TX.

Aircraft↗
PubMed1991

A double-label analysis demonstrating that all enkephalin-immunoreactive amacrine cells in the chicken retina express neurotensin immunoreactivity.

A previous study demonstrated less than 50% co-existence between the populations of enkephalin- and neurotensin-like immunoreactive amacrine cells in the chicken retina. The present study was undertaken with the intent of re-examining this relationship using a more sensitive double-label paradigm. An examination of retinal cryosections collected throughout the retina revealed that all labelled cells express both enkephalin and neurotensin-like immunoreactivity. Therefore, these results indicate the presence of a single population of chicken amacrine cells the members of which express both these neuropeptides.

Animals↗
PubMed1991

Postnatal development of ganglion cells in the rabbit retina: characterizations with AB5 and GABA antibodies.

The use of cell-specific monoclonal antibodies provides a means by which the emergence, differentiation and maturation of retinal neurons can be studied. The present study investigates the labelling of ganglion cells in the developing rabbit retina by a ganglion cell-specific monoclonal antibody, AB5(12,13). AB5 labelling of ganglion cells was observed as early as day postnatal. By 6-8 days postnatal, AB5-labelled ganglion cells had begun differentiating into the various ganglion cell subtypes observed in the adult retina. This differentiation process appeared to continue throughout the first 3 weeks postnatal. The AB5 monoclonal antibody was also used in a double-label paradigm with an anti-gamma-aminobutyric acid (GABA) polyclonal antibody to differentiate the GABAergic ganglion cells from other GABAergic elements in the retina and to study their development. GABAergic ganglion cells were first observed at 3 days postnatal and by 6 days postnatal, it was possible to observe a wide variety of GABAergic ganglion cells ranging from small cells to large alpha-type cells. The appearance of AB5 labelling in ganglion cells at relatively early stages of development suggests that the AB5 monoclonal antibody may be a useful tool for studying the development of ganglion cell structure, distribution, synaptic relationships and neurochemical specificity.

Aging↗
PubMed1991

Organization and development of horizontal cells in the goldfish retina, I: The use of monoclonal antibody AT101.

We have produced and characterized a monoclonal antibody, AT101, which selectively labels both viable and formaldehyde-fixed horizontal cell axon terminals, but not their somas or axons, of the goldfish (Carassius auratus) retina. The antigen recognized by AT101 appears to be a cell surface glycoprotein with a molecular weight of about 35,000 Daltons, and is present exclusively or predominantly in nervous tissues of all vertebrate species examined. We have used AT101 as a probe to analyze immunocytochemically the organization of horizontal cell axon terminals (HCATs) in the adult goldfish retina, and the emergence and maturation of these terminals during retinal development. Because of continued growth at the retinal margin in adult goldfish, there is a peripheral-to-central gradient in the age of cells, with the most mature in the center and the youngest in the periphery. In the center and near periphery of the adult retina, HCATs have a fusiform morphology and form a dense network in the middle and proximal part of the inner nuclear layer. In the far peripheral retina, the axon terminals appear round or ellipsoid. The retina closest to the retinal margin is devoid of AT101 staining, indicating that either HCATs are absent or the antigen recognized by AT101 is not present on HCATs at this stage. A similar sequence of changes in staining pattern is seen during development. Although AT101 staining can first be demonstrated in the larval retina at 1 month after hatching, it appears mostly as punctate structures. At a later stage, there are round or ellipsoid structures that resemble in morphology and location (in the inner nuclear layer) those found in the far peripheral adult retina. Double-labeling experiments with AT101 and antiserum against tubulin also indicate that AT101 labels the HCATs when they first appear during development. These data suggest that the emergence and maturation of HCAT is a late event in retinal development.

Animals↗
PubMed1991

General Armstrong's monuments.

During his term as surgeon general, Maj. Gen. Harry G. Armstrong was interested in the French monument commemorating the balloon flight of John Jeffries, the first American aviator, and Jean Pierre Blanchard. Maj. Gen. Armstrong proposed to visit the monument, located in a French forest, and have it renovated with appropriate multi-national ceremonies. The monument is at the landing site of the first flight across the English Channel. The authors discovered the former and current condition of the monument in Guines, France and compare that monument, of interest to Maj. Gen. Armstrong, to a new monument named for him, the newly enlarged U.S.A.F. Armstrong Laboratory.

Aerospace Medicine↗
PubMed1991

Pharmacological characteristics of high-affinity serotonin uptake systems established through gene transfer.

Inactivation of a neurotransmitter, after its stimulated release, via high-affinity uptake mechanisms is an essential regulatory step of neurotransmission in both the central and peripheral nervous systems. To initiate explorations of the molecular mechanisms and the underlying biochemical architecture of high-affinity neurotransmitter uptake systems, we have used gene transfer technology to establish and identify novel cellular models that express these systems. Human genomic DNA was transfected into mouse L-M fibroblasts and two independently arising, clonal cell lines (L-S1 and L-S2) have been identified as expressing high-affinity serotonin (5-HT) uptake systems. The 5-HT uptake characteristics of L-S1 and L-S2 are essentially comparable (in terms of Na+ dependence, temperature sensitivity, imipramine antagonizability, kinetic saturability and high affinities) and those of L-S1 have been reported previously. Furthermore, competition studies utilizing catecholamine neurotransmitters and their amino acid precursors demonstrated that these systems are highly specific for 5-HT. Several known inhibitors of high-affinity 5-HT uptake systems (including amitriptyline, desipramine, fluoxetine, imipramine, nortriptyline, tryptamine, 5-methoxytryptamine and N-acetyl 5-methoxytryptamine) were assessed in terms of their respective potencies to inhibit 5-[3H]HT uptake by L-S1 and L-S2 cells. For L-S1 cells, the rank order of inhibitor potencies is imipramine greater than amitriptyline greater than fluoxetine greater than desipramine = nortriptyline greater than tryptamine greater than 5-methoxytryptamine greater than N-acetyl-5-methoxytryptamine. For L-S2, the rank order is similar to that of L-S1 except that fluoxetine is more potent than amitriptyline.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Double-label analyses of somatostatin's coexistence with enkephalin and gamma-aminobutyric acid in amacrine cells of the chicken retina.

Double-label analyses were performed to investigate somatostatin's coexistence with either enkephalin or gamma-aminobutyric acid (GABA) in amacrine cells of the chicken retina. Double-label immunocytochemistry revealed that although some amacrine cells labelled only for somatostatin or enkephalin, approx. 81% and 85% of somatostatin-immunopositive cells in the center and periphery of the retina, respectively, were also enkephalin-immunoreactive. Somatostatin-immunocytochemistry combined with autoradiography of high-affinity [3H]GABA uptake revealed that approx. 18% of somatostatin-immunoreactive amacrine cells exhibit high-affinity uptake of [3H]GABA.

Animals↗
PubMed1990

Localization of neuropeptide-immunoreactive neurons in the human retina.

Light microscopic immunocytochemistry was utilized to localize populations of neurons in the human retina immunoreactive for the following neuroactive peptides: substance P (SP), vasoactive intestinal polypeptide (VIP), somatostatin (SOM) and LANT-6-(H-Lys-Asn-Pro-Tyr-Ile-Leu-OH), a hexapeptide which is identical to the C-terminal half of neurotensin except for the amino acid substitutions Lys/Arg and Asn/Arg. The majority of SP immunoreactive cells were amacrine cells whose pear-shaped or oval cell bodies (about 8 microns in diameter) were situated in the proximal parts of the inner nuclear layer. A small number of SP-stained somas (about 10-15 microns in diameter) were located in the ganglion cell layer and were designated as those of displaced amacrine cells. The SP-immunoreactive processes were distributed in sublamina 1, 3 and 5 with the most dense plexus being found in sublamina 3 of the inner layer. VIP-positive cell bodies (8-9 microns) were oval or pear-shaped and were situated in the innermost cell rows of inner nuclear layer. The majority of fine VIP-immunoreactive processes extended to sublamina 3 with only a few branches distributing in sublamina 1 of the inner plexiform layer. The SOM-stained cell bodies (10-11 microns) were round and were situated in the innermost cell rows of inner nuclear layer. SOM-positive processes were observed in sublamina 1 and 2 of the inner plexiform layer. The LANT-6 immunoreactive cell bodies (12-22 microns) were either oval-, round- or pyriform-shaped and were situated in ganglion cell layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Synaptic organization of neurotensin immunoreactive amacrine cells in the chicken retina.

Immunohistochemistry was utilized to investigate the light and electron microscopic localization of neurotensinlike immunoreactive (NT) amacrine cells in the chicken retina. The NT cells possess oval cell bodies (7 microns in diameter) that are located in either the second or third tier of cells from the border of the inner nuclear and inner plexiform layers. The processes of such cells extend into the inner plexiform layer where they ramify as a narrow plexus in sublamina 1 and as a broad plexus in sublaminas 3 and 4. Additionally, stained processes are observed occasionally within sublamina 5. At the ultrastructural level, NT-positive somas exhibit a rather dense and evenly distributed peroxidase reaction product throughout their cytoplasm. The nucleus of NT amacrine cells possess a round, unindented nuclear membrane. NT-immunoreactive processes in the inner plexiform layer interact synaptically only with non-NT cells. NT processes receive synaptic input mainly from the processes of amacrine cells and to a lesser degree from bipolar cells. The large majority of NT-stained varicosities form presynaptic contacts onto the processes of amacrine cells, but are also presynaptic to bipolar cell axon terminals. Moreover, each of the above synaptic relationships can be identified in each of sublaminas 1 and 3 to 4 of the inner plexiform layer. In addition, NT processes are presynaptic to processes devoid of synaptic vesicles that may originate from ganglion cells. Finally, NT processes occasionally form synaptic contacts onto somas situated in the most proximal row of the inner nuclear layer.

Animals↗