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Biomedical subjects

K M Thomas

Publications and source records attributed to K M Thomas.

At least 37 records · Page 2Linked to original sources
PubMed2000

Neurocognitive sequelae of infants of diabetic mothers.

On the basis of animal models, it was hypothesized that infants of diabetic mothers (IDMs) would be at risk for suffering damage to the hippocampus primarily because of fetal iron deficiency, chronic hypoxia, and hypoglycemia. This, in turn, may result in impairments in recognition memory at a young age. To test this model, the memory of 6-month-old IDMs and control infants was evaluated with electrophysiological (event-related potential [ERP]) and behavioral (looking time) measures. At 12 months, the Bayley Scales of Infant Development was administered. Our ERP measures showed robust evidence consistent with memory deficits in the IDMs. In contrast, the looking time measures and the Bayley exam failed to distinguish between the groups. From these results it was concluded that the ERP, but not the behavioral, measures are able to detect, in an at-risk population, deficits in recognition memory that are thought to be mediated by damage to the hippocampus.

Cognition Disorders↗
PubMed1999

Upregulation of lysyl oxidase in vascular smooth muscle cells by cAMP: role for adenosine receptor activation.

Lysyl oxidase (LO) is a key participant in the accumulation of insoluble fibers of elastin and collagen by virtue of its role in the initiation of the covalent cross-linkages between and within individual molecules comprising these fibers. In view of the essential role played by LO in the accumulation of the fibrotic components of occlusive arterial lesions in atherosclerosis, identification of the signaling molecules which can affect the expression of the LO gene in vascular smooth muscle is of considerable interest. In the present report, we describe evidence for the role of the second messenger, cAMP, in the modulation of the levels of LO in vascular smooth muscle cells. Elevated intracellular cAMP induces the transcription of the LO gene, as revealed by Northern blot analysis and nuclear run on assays. Transient transfection experiments performed with the wild-type LO promoter and with this promoter mutated at a consensus CREB site, located within the region -100 to -93 base pairs relative to the start of transcription, indicate that cAMP-induced transcriptional activation is partially due to the presence of this CREB site within the promoter. Activation of stimulatory adenosine receptors in vascular smooth muscle cells with 5'-N-ethylcarboxamido adenosine (NECA) increases cAMP, LO mRNA, and enzyme activity. These findings point to the importance of cAMP signaling, potentially initiated by a variety of physiological agents, in the upregulation of LO expression in vascular smooth muscle cells.

Adenosine-5'-(N-ethylcarboxamide)↗
PubMed1999

A developmental functional MRI study of spatial working memory.

Functional magnetic resonance imaging (fMRI) was used to examine patterns of cortical activity in children during performance of a spatial working memory task. Six children (8-10 years) and six adults (19-26 years) searched a linear array of four boxes for the appearance of a dot. In the visual blocks, participants made no response. In the motor blocks, participants were instructed to indicate the location of the dot on each trial using a button-press response. In the working memory blocks, participants were instructed to indicate at which location the dot had appeared 1 or 2 trials previously. Both children and adults showed activity in the left precentral and postcentral gyri, as well as the right cerebellum for the motor condition as compared to the visual condition. Comparison of the memory and motor conditions revealed reliable activity in the right superior frontal gyrus (BA 8), right dorsolateral prefrontal cortex (BA 10/46), right superior parietal cortex, and bilateral inferior parietal cortex for both adults and children. These results suggest that spatial working memory tasks activate very similar cortical regions for school-age children and adults. The findings differ from previous imaging studies of nonspatial working memory tasks in that the prefrontal activations observed in the current work tend to be more dorsal. Results are discussed in light of the significant behavioral performance differences observed between child and adult participants.

Adult↗
PubMed1998

Degradation and repair of elastic fibers in rat lung interstitial fibroblast cultures.

BACKGROUND: Evidence from in vitro and in vivo studies indicates that damaged elastic fibers can be repaired. METHODS: Lipid interstitial pulmonary fibroblasts were cultured for 6 weeks. Cultures were then exposed to 25 microg of porcine pancreatic elastase and fixed in pairs (control, elastase-treated) immediately after exposure and at 1, 2, 3, 4, 7, 10, 14, and 22 days for ultrastructural examination. Elastin was also analyzed biochemically for resistance to hot alkali, an indicator of repair. Steady-state levels of tropoelastin and lysyl oxidase mRNA at 2, 4, and 7 days after elastase treatment were determined by Northern blot analysis. RESULTS: Immediately after exposure to elastase, damaged elastic fibers exhibited a frayed, porous appearance and a granular texture. Through day 4, fibers showed no evidence of repair. By day 7, the granular texture of damaged fibers was no longer evident and a gradual filling-in of porous areas appeared to be taking place. By 22 days, elastic fibers were indistinguishable from elastic fibers in control cultures. The ultrastructural changes were paralleled by changes in hot alkali resistance. Through day 4, there was no change in the level of hot alkali resistant elastin. Between day 4 and day 7, resistance to hot alkali increased sharply and continued to increase at a slower rate, reaching 84% of the control level by day 22. Steady-state levels of tropoelastin and lysyl oxidase mRNA showed no increase over control levels at 2, 4, and 7 days after elastase treatment. CONCLUSIONS: Elastic fibers synthesized by lipid interstitial pulmonary fibroblasts in culture were repaired after damage by elastase. This type of repair may have relevance to the prevention of pathological conditions, such as emphysema.

Animals↗
PubMed1998

Delayed recognition memory in infants and adults as revealed by event-related potentials.

The present study sought to compare adult and infant ERP responses in analogous tests of visual recognition memory. Adults were tested under two test conditions, in which they were given either explicit instructions to respond to a previously-seen stimulus, or no instructions. Testing in both conditions took place after either a 1- or 5-min delay between familiarization and test. Eight-month-old infants were tested under the same 'passive' (no instruction) conditions as adults. ERP morphology differed across age groups. Adults evinced a P300 that was larger under 'active' as compared to 'passive' test conditions, and a late positive slow wave response that was greater to novel than familiar stimuli. There was no evidence in infants of a P300, although infants did demonstrate a late positive slow wave that was greater to familiar than to novel stimuli. Parallels between the infant and adult findings are discussed and in particular, whether the infant slow wave response is the precursor to the adult P300.

Adolescent↗
PubMed1997

Localization and activity of lysyl oxidase within nuclei of fibrogenic cells.

Lysyl oxidase (EC 1.4.3.13) oxidizes peptidyl lysine to peptidyl aldehyde residues within collagen and elastin, thus initiating formation of the covalent cross-linkages that insolubilize these extracellular proteins. Recent findings raise the possibility that this enzyme may also function intracellularly. The present study provides evidence by immunocytochemical confocal microscopy, Western blot analysis, enzyme assays, and chemical analyses for lysyl oxidase reaction products that this enzyme is present and active within rat vascular smooth muscle cell nuclei. Confocal microscopy indicates its presence within nuclei of 3T3 fibroblasts, as well.

Amino Acid Sequence↗
PubMed1997

Transcriptional and post-transcriptional control of lysyl oxidase expression in vascular smooth muscle cells: effects of TGF-beta 1 and serum deprivation.

Transforming growth factor-beta 1 (TGF-beta 1) markedly reduced cell proliferation and elevated steady state lysyl oxidase (LO) mRNA 3-fold in neonatal rat aorta smooth muscle cells cultured in medium containing 10% fetal bovine serum. The increase in LO mRNA was prevented by the presence of cycloheximide, indicative of controlling events at the level of protein synthesis. The basal level of mRNA in cells proliferating in 10% fetal bovine serum in the absence of TGF-beta 1 was enhanced 7-fold upon decreasing growth by shifting to medium containing 0.5% serum. Changes in LO activity paralleled those in LO mRNA. Nuclear run-on assays revealed that the stimulation of expression in 0.5% serum involved increased gene transcription whereas that caused by TGF-beta 1 was mostly post-transcriptional in origin. LO mRNA was quite labile (t1/2 approximately 3 h) in 10% serum but was markedly stabilized (t1/2 > 12 h) by the presence of TGF-beta 1 in the 10% serum medium. LO mRNA was also considerably more stable under retarded growth conditions (0.5% serum) in the absence of TGF-beta 1. LO promoter activity in luciferase reporter constructs transfected into these cells was low and not significantly affected by the addition of TGF-beta 1 to the 10% serum medium but was markedly elevated by shifting from 10 to 0.5% serum in the absence of TGF-beta 1. Thus, LO expression is inversely correlated with cell proliferation, and is subject to control at transcriptional and post-transcriptional levels. TGF-beta 1 enhances LO expression in these cells by dramatically stabilizing LO mRNA.

Animals↗
PubMed1997

Repair of elastase-digested elastic fibers in acellular matrices by replating with neonatal rat-lung lipid interstitial fibroblasts or other elastogenic cell types.

Disruption of elastic fibers is a major factor in the pathogenesis of pulmonary emphysema. Elastic fibers in culture, injured by exposure to elastase, undergo repair in the presence of elastogenic cells that restores the fibers toward normal as determined by biochemical and ultrastructural methods. The repair appears to be the result of both salvage and de novo repair mechanisms. The evidence for salvage repair is that hot-alkali resistance, lost as a result of elastase treatment, is restored to previously radiolabeled elastic fibers. This repair mechanism has been shown in aortic smooth muscle cell cultures. In order to determine the potential relevance of this mechanism for elastic fiber repair in the lungs, experiments were carried out using neonatal rat lung lipid interstitial fibroblasts (LIF). Treatment of the LIF cultures with elastase, in the absence of serum, caused solubilization of 12% of elastin; however, 81% of the elastin protein and 80% of the elastin-associated radioactivity (EAR) were solubilized by subsequent hot-alkali treatment, indicating that most of the elastin was retained in the matrix but was damaged. Ultrastructurally, the elastic fibers were frayed. After 6 additional wk in culture, hot-alkali resistant elastin protein and EAR were restored to 88 and 62% of control values, respectively, and the ultrastructural appearance of elastic fibers was restored to normal. We calculate that about 42% of the restored elastin represented salvage repair; the remainder was new elastin. No repair occurred in matrices rendered acellular by azide treatment; however, when acellular matrices were replated with LIF, repair was complete at 6 wk. No repair was seen when acellular matrices were replated with a transformed mouse macrophage cell line. We conclude that lung LIF are capable of mounting a robust repair process after elastolytic injury of elastin and that the repair is the result of both salvage and de novo repair mechanisms.

3T3 Cells↗
PubMed1996

Age-related changes in the electrophysiological response to visual stimulus novelty: a topographical approach.

The relationship of task relevance and stimulus probability to P300 morphology, latency and distribution was assessed. Eight year olds and adults completed visual oddball tasks of recognition memory with frequent non-target (60%), infrequent target (20%), and infrequent novel (20%) stimuli. Stimuli consisted of 2 female faces posing neutral expressions, and 40 trial unique novel photographs depicting scenes, animals, objects or abstract patterns. Event-related potentials were recorded from 17 electrodes over frontal, central and parietal scalp, including lateral temporal sites. All stimuli elicited P300 responses at parietal electrodes, with the largest responses to the target stimuli (relevant and infrequent). The P300 responses of adults and children were morphologically dissimilar, with children showing broader peaks and latency shifts across electrodes. In addition, the eight year olds displayed a frontal negativity to novel stimuli which was absent in the responses of adult participants. Results suggest that different anatomical or functional circuitry may be involved in the processing of novelty for adults as compared to eight year olds.

Adult↗
PubMed1995

Expression of lysyl oxidase from cDNA constructs in mammalian cells: the propeptide region is not essential to the folding and secretion of the functional enzyme.

Rat aortic lysyl oxidase cDNA was expressed under a metallothionein promoter in Chinese hamster ovary cells using a dihydrofolate reductase selection marker. One methotrexate-resistant cell line, LOD-06, generated by transfecting with full-length cDNA, yielded lysyl oxidase proteins consistent with the 50 kDa proenzyme and a 29 kDa mature catalyst. A second cell line, LOD32-2, was generated by transfection with a truncated cDNA lacking sequences which code for the bulk of the propeptide region. Both cell lines secreted apparently identical, 29 kDa forms of mature lysyl oxidase each of which catalyzed the deamination of human recombinant tropoelastin and alkylamines, consistent with the known specificity of lysyl oxidase. The secreted enzyme forms were inhibited by chemical inhibitors of lysyl oxidase activity, including beta-aminopropionitrile, phenylhydrazine, ethylenediamine, alpha, alpha'-dipyridyl, and diethyldithiocarbamate. Sensitivity to these agents is consistent with the presence of copper and carbonyl cofactors in the expressed enzymes, characteristic of lysyl oxidase from connective tissues. These results indicate the lack of essentiality of the deleted proprotein sequence for the proper folding, generation of catalytic function, and secretion of lysyl oxidase.

Amino Acid Sequence↗
PubMed1994

Molecular characterization of a novel rabbit interleukin-8 receptor isotype.

Interleukin-8 (IL-8) mediates the transendothelial migration and activation of neutrophils to the site of inflammation. Two human IL-8 receptor isotype (A and B) and one rabbit IL-8 receptor isotype (A) cDNAs have been previously cloned and characterized on the basis of their pharmacological profile. Human and rabbit IL-8 receptor subtype A binds IL-8 and structurally related peptide melanoma growth-stimulating activity (MGSA) and neutrophil-activating peptide-2 (NAP-2) according to the following affinity binding profile: IL-8 >>> MGSA > NAP-2, whereas the human IL-8 receptor subtype B profile is IL-8 = MGSA > NAP-2 (LaRosa, G., Thomas, K. M., Kaufmann, M., Mark, R., White, M., Taylor, L., Gray, G., Witt, D., and Navarro, J. (1992) J. Biol. Chem. 267, 25402-25406). In this study, we isolated a cDNA clone (5B1a) from a rabbit neutrophil library encoding a G-protein-coupled receptor of the interleukin-8 receptor family. The 5B1a clone encodes a 358-amino acid protein exhibiting 80% amino acid identity to the human IL-8 receptor B, 74% to the rabbit IL-8 receptor A, and 73% to the human IL-8 receptor A. Tissue distribution by Northern blot analysis reveals that the 5B1a mRNA is expressed preferentially in neutrophils. In contrast to previously described IL-8 receptors, the 5B1a receptor exhibited specific 125I-IL-8 binding with a novel affinity binding profile of IL-8 >> NAP-2 > MGSA. The corresponding apparent Ki values for IL-8, NAP-2, and MGSA were 4, 120, and 320 nM, respectively. IL-8 induced intracellular calcium mobilization and desensitization in Chinese hamster ovary cells stably transfected with 5B1a, indicating that this cDNA encodes a functional IL-8 receptor. Sequence analysis of the 5B1a protein with other IL-8 receptor subtypes within the framework of their pharmacological profile reveals putative structural motifs that may correspond to the ligand binding site of the IL-8 receptor.

Amino Acid Sequence↗
PubMed1994

Functional and ligand binding specificity of the rabbit neutrophil IL-8 receptor.

IL-8 mediates migration and activation of neutrophils. This study describes the functional and ligand binding specificity of the human intercrine peptides IL-8, neutrophil-activating peptide 2 (NAP-2), melanoma growth stimulatory activity (GRO), and platelet factor 4 (PF4) to rabbit neutrophils and mammalian cell lines transfected with rabbit IL-8 receptor cDNA (F3R). Rabbit neutrophil membranes bound 125I-labeled IL-8 and 125I-labeled NAP-2 but did not bind 125I-labeled GRO or 125I-labeled PF4. Rabbit neutrophils mobilized intracellular Ca2+ in response to IL-8 and NAP-2 but not to GRO or PF4. Monkey kidney cells (COS-7) and hamster lung fibroblasts (CCL-39) were transiently and stably transfected with the rabbit neutrophil IL-8 receptor F3R cDNA. COS-7 cells transfected with F3R cDNA bound 125I-labeled IL-8 but did not bind other IL-8-related peptides such as 125I-labeled NAP-2, 125I-labeled GRO, or 125I-labeled PF4. Furthermore, bound 125I-labeled IL-8 was only displaced by unlabeled IL-8 but not by unlabeled NAP-2, GRO alpha, or PF4. Consistent with this observation, stably transfected CCL 39 cells expressing F3R cDNA mobilized Ca2+ only in response to IL-8. We conclude that F3R cDNA encodes a functional IL-8 receptor isotype with strict ligand binding specificity for IL-8, that rabbit neutrophils do not bind human GRO alpha, and it is suggested that rabbit neutrophils contain in addition to the F3R protein another IL-8 receptor isotype with broad ligand specificity or a distinct NAP-2 receptor.

Animals↗
PubMed1993

The rabbit neutrophil N-formyl peptide receptor. cDNA cloning, expression, and structure/function implications.

The rabbit neutrophil N-formyl peptide receptor (FPR) has been well studied for its ligand binding properties. Recent gene cloning experiments have established the existence of a subfamily of G protein-coupled receptors that share extensive sequence homology with the FPR, yet lack the capability of high affinity binding to FMLP. These findings prompted us to identify the structural requirement for formyl peptide ligand binding by delineation of the primary structure of the rabbit FPR. A rabbit neutrophil cDNA library was screened with a cloned human FPR cDNA probe and the insert of one positive isolate (B6) was sequenced. The 1268-bp cDNA insert encodes a peptide of 352 amino acids. Stably transfected L cell fibroblasts expressing the rabbit cDNA displayed specific binding of the ligand fMet-Leu-[3H]Phe with two affinities (Kd = 0.31 and 7.5 nM). Addition of the nonhydrolyzable guanosine triphosphate analogue, GTP gamma S, converted > or = 85% of the high affinity sites to the low affinity sites. FMLP induced mobilization of intracellular calcium in the transfected cells (EC50 = 0.5 nM), a response sensitive to pertussis toxin. FMLP stimulation desensitized the receptor such that subsequent stimulation with the same ligand produced a significantly reduced signal. These results indicate that the cloned rabbit receptor represents a high affinity FPR, and that FPR-mediated early signal transduction events can be fully reconstituted in transfected mammalian cells. The rabbit FPR sequence is 78% identical to that of the human FPR, and 68% identical to FPR2, a homologue of FPR with a low binding affinity (Kd > or = 400 nM) for FMLP. Analysis of the aligned sequences of these three proteins revealed that: 1) the amino termini and the second extracellular loops have the lowest sequence homology; 2) sequence in the intracellular domains that couple to G protein are highly conserved; and 3) the first and the third extracellular loops and their adjacent transmembrane domains of the FPR may contain residues essential for the high affinity binding of FMLP.

Amino Acid Sequence↗
PubMed1992

Amino terminus of the interleukin-8 receptor is a major determinant of receptor subtype specificity.

Interleukin-8 (IL-8) is a key mediator in the migration of neutrophils from the circulation to the site of inflammation in the tissue. IL-8 is secreted by many cell types in response to proinflammatory stimuli such as interleukin 1, tumor necrosis factor, and lipopolysaccharide and is a potent chemoattractant and activator of neutrophils. Neutrophil activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA/GRO) are structurally and functionally related to IL-8 and, like IL-8, bind to specific G protein-coupled receptors on neutrophils. In the present study two closely related cloned IL-8 receptor subtypes are characterized by expression of the cDNA clones in monkey kidney cells (COS-7) or chinese hamster ovary cells and analysis of their ligand binding profiles. Both receptor subtypes bind 125I-labeled IL-8 with similar high affinity, however, the F3R receptor binds IL-8 exclusively, while the 4Ab receptor binds both IL-8 and MGSA/GRO with high affinity and NAP-2 with lesser affinity. Furthermore, we demonstrate with the use of intersubtype chimeric receptors that the specificity of ligand binding to both IL-8 receptor subtypes is dictated by the heterogeneous NH2-terminal domain. The F3R receptor is representative of a restricted IL-8 receptor subtype, and 4Ab represents a nonrestricted receptor subtype. It is proposed that these subtypes be named IL-8 receptors alpha and beta, respectively.

Amino Acid Sequence↗
PubMed1991

The interleukin-8 receptor is encoded by a neutrophil-specific cDNA clone, F3R.

Interleukin-8 (IL-8) is one of the most potent chemotactic agents for neutrophils and has been implicated as a major mediator of inflammation. The IL-8 receptor is expressed exclusively in neutrophils and belongs to the family of G-protein-coupled receptors. In a recent paper we reported the characterization of a cDNA clone, F3R, isolated from a neutrophil cDNA library and showed that it encodes a G-protein-coupled receptor which is exclusively expressed in neutrophils. We also suggested, based on expression studies in Xenopus oocytes, that the F3R protein product is an isoform of the (fMLP) receptor (Thomas, K. M., Pyun, H. Y., and Navarro, J. (1990) J. Biol. Chem. 265, 20061-20064). In this work, the F3R receptor cDNA is expressed in monkey kidney cells (COS-7) and is shown to encode the IL-8 receptor. F3R cDNA does not encode for a fMLP receptor isoform. We show conclusively that the F3R-transfected COS-7 cells express the IL-8 receptor at a density equivalent to that observed in neutrophils. The pharmacological profile of the F3R-transfected cells is the same as that of neutrophils. The apparent Kd values for binding of 125I-IL-8 to neutrophils and F3R-transfected COS-7 cell membranes were 1.2 and 1.4 nM, respectively. Antipeptide antibodies against a partial sequence of the F3R protein product specifically immunoprecipitate the IL-8 receptor from transfected cells as well as neutrophils. The molecular characterization of the IL-8 receptor should provide the basis for further studies on the identification of the binding domain of this inflammatory receptor.

Animals↗