Biomedical subjects

T Lee

Publications and source records attributed to T Lee.

At least 127 records · Page 7Linked to original sources

PubMed1999

Purification and characterization from rat kidney membranes of a novel platelet-activating factor (PAF)-dependent transacetylase that catalyzes the hydrolysis of PAF, formation of PAF analogs, and C2-ceramide.

We have previously identified two enzyme activities that transfer the acetyl group from platelet-activating factor (PAF) in a CoA-independent manner to lysoplasmalogen or sphingosine in HL-60 cells, endothelial cells, and a variety of rat tissues. These were termed as PAF:lysoplasmalogen (lysophospholipid) transacetylase and PAF:sphingosine transacetylase, respectively. In the present study, we have solubilized and purified this PAF-dependent transacetylase 13,700-fold from rat kidney membranes (mitochondrial plus microsomal membranes) based on the PAF:lysoplasmalogen transacetylase activity. The mitochondria and microsomes were prepared and washed three times, then solubilized with 0.04% Tween 20 at a detergent/protein (w/w) ratio of 0.1. The solubilized fractions from mitochondria and microsomes were combined and subjected to sequential column chromatographies on DEAE-Sepharose, hydroxyapatite, phenyl-Sepharose, and chromatofocusing. The enzyme was further purified by native-polyacrylamide gel electrophoresis (PAGE) and affinity gel matrix in which the competitive inhibitor of the enzyme, 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphoethanolamine was covalently attached to the CH-Sepharose. On SDS-PAGE, the purified enzyme showed a single homogeneous band with an apparent molecular mass of 40 kDa. The purified enzyme catalyzed transacetylation of the acetyl group not only from PAF to lysoplasmalogen forming plasmalogen analogs of PAF, but also to sphingosine producing N-acetylsphingosine (C2-ceramide). In addition, this enzyme acted as a PAF-acetylhydrolase in the absence of lipid acceptor molecules. These results suggest that PAF-dependent transacetylase is an enzyme that modifies the cellular functions of PAF through generation of other diverse lipid mediators.

Acetyltransferases↗

PubMed1999

Induction of P-glycoprotein expression in astrocytes following intracerebroventricular kainate injections.

The expression of P-glycoprotein (PGP) was studied by immunocytochemistry and light and electron microscopy, in normal rats and after intracerebroventricular kainate injections. Two antibodies to PGP, mdr (Ab-1) and c-219, were used. As in previous studies (Thiebault et al. and Jette et al.), labelled capillaries were observed in normal rats. Kainate injections resulted in death of pyramidal neurons in the hippocampus, and a proliferation of glial cells in the affected cornu ammonis fields. An increase in PGP expression was observed in reactive astrocytes as early as 1 day postinjection. Immunoreactivity peaked at 2 weeks postinjection, but was still visible as late as 10 weeks postinjection. Similar results were observed using the two antibodies. Double immunolabelling and confocal microscopy also showed that PGP was colocalised with GFAP, a marker for astrocytes. The expression of PGP in astrocytes was confirmed by electron microscopy, which showed immunoreaction product in cells containing dense bundles of glial filaments and features of reactive astrocytes. The increased PGP expression in reactive astrocytes could be part of a cellular stress response program in these cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Time-course and localization of transferrin receptor expression in the substantia nigra of 6-hydroxydopamine-induced parkinsonian rats.

Parkinson's disease is a neurodegenerative disease characterized by dopaminergic cell death in the substantia nigra. The cause of the cell death is, however, obscure. Recently, accumulation of iron in the parkinsonian substantia nigra and iron-catalysed free radical generation have been proposed as possible causes of nigral cell death. The transferrin receptor has been implicated as a possible mediator of this iron accumulation in the parkinsonian substantia nigra. The present study investigated the distribution of transferrin receptor-immunoreactive proteins and its co-localization with tyrosine hydroxylase in the normal rat substantia nigra and their expressions in the parkinsonian substantia nigra from three days to three months after 6-hydroxydopamine lesioning. Computer image analysis of the grey mean of transferrin receptor staining in the microvessels was also employed. The results showed that the transferrin receptor immunolabelling was localized in some neurons and glial cells in the normal substantia nigra pars compacta and pars reticulata, and that about 54% of tyrosine hydroxylase-positive cells were also stained with transferrin receptor. There was a decrease of tyrosine hydroxylase- and transferrin receptor-positive cells in the 6-hydroxydopamine-lesioned substantia nigra. The grey mean of transferrin receptor staining in microvessels in the lesioned substantia nigra was, however, not different from that in the control. It was concluded that transferrin receptors in neurons, glial cells and microvessels might not be responsible for iron accumulation in the parkinsonian substantia nigra. The loss of transferrin receptor-immunopositive cells might, however, partly be accounted for by the death of transferrin receptor-positive dopaminergic cells induced by 6-hydroxydopamine lesioning.

Animals↗

PubMed1999

Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.

We describe a genetic mosaic system in Drosophila, in which a dominant repressor of a cell marker is placed in trans to a mutant gene of interest. Mitotic recombination events between homologous chromosomes generate homozygous mutant cells, which are exclusively labeled due to loss of the repressor. Using this system, we are able to visualize axonal projections and dendritic elaboration in large neuroblast clones and single neuron clones with a membrane-targeted GFP marker. This new method allows for the study of gene functions in neuroblast proliferation, axon guidance, and dendritic elaboration in the complex central nervous system. As an example, we show that the short stop gene is required in mushroom body neurons for the extension and guidance of their axons.

Animals↗

PubMed1999

Controlled release of human immunoglobulin G. 1. Release kinetics studies.

The release of human immunoglobulin G (IgG) using ethylene-vinyl acetate copolymer (EVAc) as a polymer carrier was studied by fabricating them into two commercially available dosage forms: slab and microsphere. A first-order flux decay model and two hierarchical models concerned with the mass transfer coefficient on the slab surface were used to describe the mechanism of release kinetics and the results compared. These models gave insight to some of the important physical parameters of drug release such as the diffusion coefficient, time constant of release, and initial flux. It was found that the release mechanism varies with time, and hence no single model can be used to predict the release profile for the entire period of study. A controlled release study by matrix coating was also done. The results obtained were utilized to examine the suitability of a particular dosage form (matrix geometry) of IgG for clinical applications. The release data compared with the standard methods of IgG therapy proves localized drug delivery to be a major boon for immunodeficient patients.

Algorithms↗

PubMed1999

Controlled release of human immunoglobulin G. 2. Morphological characterization.

Human immunoglobulin G (IgG) serves as an important chemotherapeutic agent for a number of immunological ailments and as a carrier in the targeted delivery of other therapeutic agents. This paper deals with the characterization of IgG-dispersed monolithic matrixes of different geometries, prepared using a nonbiodegradable polymer carrier EVAc. The morphological changes associated with the matrix during drug release was studied using scanning electron microscopy, polarizing microscopy, atomic force microscopy, and X-ray photoelectron microscopy, and the results were compared. The study answered the burst effect problem significantly and illustrated the potential of these techniques in understanding the morphological structure of matrixes and mode of release kinetics.

Delayed-Action Preparations↗

PubMed1999

Students' part-time work: towards an understanding of the implications for nurse education.

This paper reports on the subject of nursing students undertaking paid part-time work, an area which has attracted minimal research. A questionnaire survey of 120 diploma students in one university investigated several aspects of part-time work: incidence, nature, motivating factors, and effects on their professional and personal lives. The results provide an insight into the work profile and the students' perceptions of the positive and negative effects on their personal well being and progression on the course. The findings raise issues which are of interest to both academic and clinical staff, nursing students and employers.

Adolescent↗

PubMed1999

Time course of dopaminergic cell death and changes in iron, ferritin and transferrin levels in the rat substantia nigra after 6-hydroxydopamine (6-OHDA) lesioning.

Parkinson's disease is characterized by dopaminergic cell death in the substantia nigra. The underlying mechanism is, however, unknown. Though there are increasing lines of evidence showing iron accumulation in the Parkinsonian substantia nigra, it still remains obscure whether increased iron is the primary cause of dopaminergic cell death, or just a consequence of the pathological process. It is also unclear how iron gains access to the Parkinsonian SN. To gain more understanding in these areas, the present study investigated the time course of dopaminergic cell death and of changes in the level of iron, ferritin and transferrin. The results showed that iron was increased after the significant nigral dopaminergic cell death induced by 6-hydroxydopamine injection into the rat substantia nigra. On the other hand, the expression of transferrin was decreased. However, there was a temporal increase in the number of ferritin positive microglia. The results indicated that iron increase was not the primary cause of dopaminergic cell death in the Parkinsonian rat. It was most likely the result of an accumulation of iron-laden microglia.

Animals↗

PubMed1999

Hypoxia upregulates VEGF production in keloid fibroblasts.

The etiology of keloid formation is diverse. They are characterized grossly as thick scar tissue that extends beyond the boundaries of the original wound. Histologically, keloids are composed of excessive collagen with an abnormally large number of partially or totally occluded microvessels. This occlusion of keloid microvessels has been hypothesized to contribute to a hypoxic microenvironment within these pathological scars. Vascular endothelial growth factor (VEGF), a potent endothelial cell mitogen, and proangiogenic cytokine have been implicated in normal and pathological wound healing. The purpose of this study was to evaluate the amount of VEGF protein production by fibroblast cell lines derived from keloids and normal human dermal skin in hypoxic compared with normoxic culture conditions. By enzyme-linked immunosorbent protein assay, VEGF was increased in both keloid and normal human dermal fibroblasts in hypoxia over normoxic controls. There was not, however, a significant difference between upregulation of VEGF protein when comparing the keloid and normal fibroblast groups. As the result of the data, alternative hypotheses for hypoxia-induced keloid formation were explored: (1) downstream modulation or signal transduction of VEGF, (2) VEGF production from cells other than fibroblasts, (3) the importance of matrix accumulation stimulated by hypoxia, or (4) increased migration of cells (other than fibroblasts) specific to keloid biology. These hypotheses may help explain the possible role of hypoxia in the pathogenesis of keloid formation. Future studies involving in situ hybridization or immunohistochemical analysis may offer greater insight into the mechanisms underlying keloid formation. Ultimately, our therapeutic goal is the utilization of biomolecular approaches for the suppression of keloid formation.

Endothelial Growth Factors↗

PubMed1999

Pathological difference between retractile and cryptorchid testes.

PURPOSE: We compared testicular biopsies from retractile and cryptorchid testes to determine the histological effect of testicular retraction and the necessity of treatment for retractile testes. MATERIALS AND METHODS: A total of 61 testicular biopsies were performed during orchiopexy in 36 boys 1.3 to 9.8 years old (mean age 5.4) with retractile testes (unilateral in 11, bilateral in 50) and 115 testicular biopsies were done in 83 patients with cryptorchidism (unilateral in 51, bilateral in 64) 0.5 to 14.9 years old (mean age 3.7). Parameters for germ and Sertoli cells were determined in each group. RESULTS: Mean average spermatogonial number (S/T value) and Sertoli cell index were statistically different between retractile and cryptorchid testes with values of 2.96+/-1.33 versus 0.61+/-0.87 and 26.81+/-6.75 versus 23.04+/-5.85, respectively. Average tubular degeneration phase V to VII ratio was 0.23+/-0.18 for retractile testes and 0.22+/-0.17 for cryptorchid testes which was not statistically different. CONCLUSIONS: Similar tubular degeneration phase V to VII values between retractile and cryptorchid testes indicate histological change in retractile testes and suggest the need for hormonal or surgical therapy for those patients with retractile testes lacking spontaneous descent.

Biopsy↗

PubMed1999

Lymphatic absorption of structured triglycerides vs. physical mix in a rat model of fat malabsorption.

Comparison was made between the intestinal absorption and lymphatic transport of a randomly interesterified fish oil and medium-chain triglyceride (MCT) structured triglycerides (STG) vs. the physical mix in rat small intestine following ischemia and reperfusion (I/R) injury. Under halothane anesthesia, the superior mesenteric artery (SMA) was occluded for 20 min and then reperfused in I/R rats. The SMA was isolated but not occluded in control rats. In both treatment groups, the mesenteric lymph duct was cannulated and a gastric tube was inserted. Each treatment group received 1 ml of the fish oil-MCT STG or physical mix (7 rats/group) through the gastric tube followed by an infusion of PBS at 3 ml/h for 8 h. Lymph was collected hourly for 8 h. Lymph triglyceride, cholesterol, and decanoic and eicosapentaenoic acids increased rapidly and maintained a significantly higher output (P < 0.01) with STG compared with physical mix in control rats over 8 h. After I/R, lymphatic triglyceride output decreased 50% compared with control. Gastric infusion of STG significantly improved lipid transport by having a twofold higher triglyceride, cholesterol, and decanoic and eicosapentaenoic acids output to lymph compared with its physical mix (P < 0.01). We conclude that STG is absorbed into lymph significantly better than physical mix by both the normal intestine and the intestine injured by I/R.

Animals↗

PubMed1999

Characterization of MAP kinase and PKC isoform and effect of ACE inhibition in hypertrophy in vivo.

Protein kinase C (PKC) and mitogen-activated protein (MAP) kinase activation appear important in conferring hypertrophy in vitro. However, the response of PKC and MAP kinase to stimuli known to induce hypertrophy in vivo has not been determined. We recently demonstrated that pressure-overload hypertrophy induced a transiently transfected gene driven by an hypertrophy responsive enhancer (HRE) through a marked increase in binding activity of its interacting nuclear factor (HRF). These data suggested that the HRE/HRF could serve as a target for evaluating the signal transduction events responsible for hypertrophy in vivo. Accordingly, we characterized MAP kinase and PKC isoform activation, injected HRE driven reporter gene expression, and HRF binding activity in rat hearts subjected to ascending aortic clipping or sham operation in the presence of the angiotensin-converting enzyme (ACE) inhibitor fosinopril, hydralazine, or no treatment. Analyses showed that PKC-epsilon and MAP kinase were acutely activated following ascending aortic ligature and that fosinopril significantly inhibited but did not completely abrogate PKC-epsilon and MAP kinase activation. However, fosinopril completely prevented pressure overload-mediated induction of HRE containing constructs and obviated increased HRF binding activity. These results suggest a direct relationship between ACE activity and HRE/HRF-mediated gene activation and imply that PKC-epsilon and MAP kinase may be involved in transducing this signal.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1999

Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast.

The mushroom bodies (MBs) are prominent structures in the Drosophila brain that are essential for olfactory learning and memory. Characterization of the development and projection patterns of individual MB neurons will be important for elucidating their functions. Using mosaic analysis with a repressible cell marker (Lee, T. and Luo, L. (1999) Neuron 22, 451-461), we have positively marked the axons and dendrites of multicellular and single-cell mushroom body clones at specific developmental stages. Systematic clonal analysis demonstrates that a single mushroom body neuroblast sequentially generates at least three types of morphologically distinct neurons. Neurons projecting into the (gamma) lobe of the adult MB are born first, prior to the mid-3rd instar larval stage. Neurons projecting into the alpha' and beta' lobes are born between the mid-3rd instar larval stage and puparium formation. Finally, neurons projecting into the alpha and beta lobes are born after puparium formation. Visualization of individual MB neurons has also revealed how different neurons acquire their characteristic axon projections. During the larval stage, axons of all MB neurons bifurcate into both the dorsal and medial lobes. Shortly after puparium formation, larval MB neurons are selectively pruned according to birthdays. Degeneration of axon branches makes early-born gamma neurons retain only their main processes in the peduncle, which then project into the adult gamma lobe without bifurcation. In contrast, the basic axon projections of the later-born (alpha'/beta') larval neurons are preserved during metamorphosis. This study illustrates the cellular organization of mushroom bodies and the development of different MB neurons at the single cell level. It allows for future studies on the molecular mechanisms of mushroom body development.

Animals↗

PubMed1998

Regulation of the neutrophil respiratory burst oxidase. Identification of an activation domain in p67(phox).

Superoxide generation by the neutrophil respiratory burst oxidase (NADPH oxidase) can be reconstituted in a cell-free system using flavocytochrome b558 and the cytosolic proteins p47(phox), p67(phox), and Rac. p47(phox) functions as an adaptor protein; it increases the affinity of p67(phox) and Rac in the NADPH oxidase complex, but is not essential when high concentrations of these proteins are used (Freeman, J. L., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 22578-22582), implying that p67(phox) and/or Rac directly regulates enzyme activity. Herein, we describe an activation domain in p67(phox) that is essential for NADPH oxidase activity. A series of C-terminal truncation mutants of p67(phox) showed that residues 211 to the C terminus (residue 526) are not needed for cell-free activity. However, shorter truncations were inactive, pointing to an activation domain within the region spanning residues 199-210. p67(phox) mutated at single amino acid residues within this region showed diminished activity, and p67(phox) V204A was completely inactive. The effects of mutations on activity were independent of p47(phox), and mutations did not affect the binding of p67(phox) to Rac. In the presence of wild-type p67(phox), the V204A mutant was a potent inhibitor of superoxide generation, and inhibition was partially reversed by high concentrations of p67(phox), but not by p47(phox) or Rac. The V204A mutant competed with native p67(phox) for translocation to neutrophil plasma membrane, indicating that p67(phox) V204A assembles to form an inactive complex. The data imply a direct activation of flavocytochrome b558 by an activation domain in p67(phox).

Amino Acid Sequence↗

PubMed1998

Influence of promoter potency on the transcriptional effects of YY1, SRF and Msx-1 in transient transfection analysis.

Potent viral promoters/enhancers are often used to achieve high level expression of ectopic genes in transient transfection analysis. By using a GAL4-responsive transcription assay system, we show that the use of potent eukaryotic expression vectors can lead to biased transcriptional effects. Three functionally diverse transcription factors, YY1, SRF and Msx-1, were examined and each was found to exhibit a strong transrepression function in the context of the DNA binding domain of GAL4 when expressed from the cytomegalovirus (pCMV) or simian virus 40 (pSV) promoters/enhancers. An internal 15 amino acid domain of YY1 mediating transrepression in the viral promoter setting was identified. This GAL4-mediated transcriptional repression could, however, be completely relieved by using the yeast alcohol dehydrogenase promoter (pADH) to drive gene expression, which is approximately 100-fold weaker than canonical pCMV and pSV in cultured mammalian cells. In addition, low level expression achieved with the pADH vector unveiled the intrinsic transactivation functions of YY1 and SRF previously not observed with the GAL4 assay system. Our results highlight a potential pitfall in conventional pCMV- and pSV-based transfection assays and suggest that the use of a low level expression system may be preferable in most transcriptional analysis.

Alcohol Dehydrogenase↗

PubMed1998

Th1- and Th2-like cytokines in CD4+ and CD8+ T cells in autism.

Th1-like (IL-2, IFN-gamma) and Th2-like (IL-4, IL-6, and IL-10) cytokines were examined in CD4+ and CD8+ T cells in children with autism. Intracellular cytokines were measured using specific antibodies to various cytokines and anti-CD4 or anti-CD8 monoclonal antibodies by FACScan. Proportions of IFN-gamma+CD4+ T cells and IL-2+CD4+ T cells (Th1), and IFN-gamma+CD8+ and IL-2+CD8+ T cells (TC1) were significantly lower in autistic children as compared to healthy controls. In contrast, IL-4+CD4+ T cells (Th2) and IL-4+CD8+ T cells (TC2) were significantly increased in autism. The proportions of IL-6+ CD4+, IL-6+CD8+ and IL-10+CD4+, IL-10+CD8+ T cells were comparable in autism and control group. These data suggest that an imbalance of Th1- and Th2-like cytokines in autism may play a role in the pathogenesis of autism.

Autistic Disorder↗

PubMed1998

Proteolytic regulation of the zinc finger transcription factor YY1, a repressor of muscle-restricted gene expression.

Regulated proteolysis has been postulated to be critical for proper control of cell functions. Muscle development, in particular, involves a great deal of structural adaptation and remodeling mediated by proteases. The transcription factor YY1 represses muscle-restricted expression of the sarcomeric alpha-actin genes. Consistent with this repressor function of YY1, the nuclear regulator is down-regulated at the protein level during skeletal as well as cardiac muscle cell differentiation. However, the YY1 message remains relatively unaltered throughout the myoblast-myotube transition, implicating a post-translational regulatory mechanism. We show that YY1 can be a substrate for cleavage by the calcium-activated neutral protease calpain II (m-calpain) and the 26 S proteasome. The calcium ionophore A23187 destabilized YY1 in cultured myoblasts, and the decrease in YY1 protein levels could be prevented by calpain inhibitor II and calpeptin. Treatment with the proteasome inhibitors MG132 and lactacystin resulted in the stabilization of YY1 protein, which is consistent with the finding that YY1 is readily polyubiquitinated in reticulocyte lysates. We further show that proteolytic targeting by calpain II and the proteasome involves different structural elements of YY1. This study thus illustrates two proteolytic pathways through which the transcriptional regulator can be differentially targeted under different cell growth conditions.

Animals↗

PubMed1998

Analysis of the S3 and S3' subsite specificities of feline immunodeficiency virus (FIV) protease: development of a broad-based protease inhibitor efficacious against FIV, SIV, and HIV in vitro and ex vivo.

The S3 and S3' subsite binding specificities of HIV and feline immunodeficiency virus proteases (FIV) proteases (PRs) have been explored by using C2-symmetric competitive inhibitors. The inhibitors evaluated contained (1S, 2R, 3R, 4S)-1,4-diamino-1, 4-dibenzyl-2,3-diol as P1 and P1' units, Val as P2 and P2' residues, and a variety of amino acids at the P3 and P3' positions. All inhibitors showed very high potency against HIV PR in vitro, and their Ki values ranged between 1.1 and 2.6 nM. In contrast to the low restriction of P3 and P3' residues observed in HIV PR, FIV PR exhibited strong preference for small hydrophobic groups at the S3 and S3' subsites. Within this series, the most effective inhibitor against FIV PR contained Ala at P3 and P3'. Its Ki of 41 nM was 415- and 170-fold lower than those of the inhibitors without the P3 and P3' moieties or with the Phe at these positions, respectively. In addition, these compounds were tested against mutant FIV PRs, which contain amino acid substitutions corresponding to those in native HIV PR at homologous sites, and their efficacy of inhibition progressively increased up to 5-fold. The most potent FIV PR inhibitor was selected for examination of its effectiveness in tissue culture, and it was able to block nearly 100% of virus production in an acute infection at 1 microg/ml (1.1 microM) against HIV, FIV, and simian immunodeficiency virus. Furthermore, it was not toxic to cells, and even after 2 months of culture there was no sign of resistance development by virus. The findings suggest that inhibitors with small P3 residue may be efficacious against a broad range of HIV variants as well as interspecies PRs.

Animals↗