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Biomedical subjects

M Y Lee

Publications and source records attributed to M Y Lee.

At least 55 records · Page 3Linked to original sources

Craniofacial surgical simulation: application of three-dimensional medical imaging and rapid prototyping models.

BACKGROUND: Adequate assessment of the deformity, formulation of good treatment planning, and sufficient rehearsal of procedures before actually performing surgery ensure successful craniofacial surgery. Three-dimensional computed tomography (CT) imaging and facsimile models were used in combination to evaluate their function in craniofacial surgery. METHODS: Three-dimensional imaging and facsimile models were used for reconstruction of craniofacial deformity. CT data were acquired, processed, and reconstructed to display 3-dimensional images. The images were used for evaluation of the deformity. The images were then manipulated to create multiple osseous objects. Surgical simulation was performed by moving the computer images. The image processing and manipulation were achieved using the AnalyzePC program. The raw CT data were transformed into a readable format and transferred to produce facsimile models using rapid prototyping technology. The skull models were used for evaluation and surgical simulation. Both methods were compared and used to assist in surgery, which was performed according to the simulations. RESULTS: Three-dimensional CT imaging and facsimile models were helpful for simulation of craniofacial surgery. The actual surgery results were satisfactory without complications. Particular advantages were the unlimited trials with the imaging method, and the feeling of reality with the model method. CONCLUSIONS: Craniofacial surgery is facilitated by preoperative simulation of procedures. Both 3-dimensional CT imaging and facsimile models are helpful for craniofacial surgical simulation.

Craniofacial Abnormalities↗

Up-regulated CNTF plays a protective role for retrograde degeneration in the axotomized rat retina.

Using reverse transcription-polymerase chain reaction and in situ hybridization, we investigated the expression and cellular localization of ciliary neurotrophic factor receptor alpha (CNTFRalpha) in the rat retina following optic nerve transection (ONT). Following ONT, a signal for CNTFRalpha mRNA appeared in a layer-specific and time-dependent manner. In the ganglion cell layer, the signal showed a peak value 1 day after ONT, and then gradually decreased. In the inner nuclear layer the signal reached a peak value at 14 days of about 500% of control level, but then decreased at 4 weeks. Our findings suggest that CNTF might play a protective role for the retrograde degeneration of retinal cells induced by ganglion cell death in the rat retina following ONT.

Animals↗

Interaction of the retinoblastoma protein (pRb) with the catalytic subunit of DNA polymerase delta (p125).

The retinoblastoma gene product (pRb) interacts with many cellular proteins to function in the control of cell division, differentiation, and apoptosis. Several pRb binding proteins complex with pRb through an amino acid sequence called the LXCXE motif. The catalytic subunit of DNA polymerase delta (p125) contains a LXCXE motif. To further study the biochemical function of this polymerase, we sought to determine if p125 interacts with pRb. Experiments using GST-pRb fusion proteins showed that p125 from breast epithelial (MCF10A) cell extracts associates with pRb. In addition, GST-p125 fusion proteins bound pRb from the same cell extracts. The pRb that associated with GST-p125 was largely unphosphorylated. Coimmunoprecipitation experiments using cell cycle synchronized cells revealed that p125 and pRb form a complex predominantly during G1 phase, the phase during which pRb is mostly unphosphorylated. In vitro phosphorylation of GST-pRb by the cyclin dependent kinases reduced the ability of p125 to associate with GST-pRh. Addition of the LXCXE containing protein SV40 large T antigen to GST-pRb blocks the ability of p125 to associate with pRb, suggesting that it may be through a LXCXE sequence by which p125 interacts with pRb. Finally, in vitro polymerase assays demonstrate that GST-pRb fusion protein stimulates DNA polymerase delta activity.

Animals↗

Direct binding of hepatitis B virus X protein and retinoid X receptor contributes to phosphoenolpyruvate carboxykinase gene transactivation.

The X gene product of the human hepatitis B virus (HBx), a major factor responsible for hepatitis and hepatocellular carcinoma, modulates transactivation by a variety of transcription factors. Herein, expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene was found to be regulated transcriptionally by HBx through two distinct promoter regions. The cAMP response element (CRE)-1 site within the proximal promoter region mediated the HBx-induced transactivation of the PEPCK gene through C/EBP alpha and ATF-2. A retinoid X receptor (RXR) response element within the distal promoter region also contributed to the HBx-induced transactivation. Consistent with these results, HBx directly interacted with RXR, and the interaction interfaces were localized to the transactivation domain of HBx and the ligand binding domain of RXR.

Activating Transcription Factor 2↗

Expression of brain/kidney protein in Müller cells of rat retina following transient ischemia.

We have investigated the expression and cellular localization of brain/kidney (B/K) protein in the rat retina following transient ischemia. In the normal retina, strong B/K immunoreactivity was localized to some ganglion cells. In addition, a few radial Muller cell processes showed B/K immunoreactivity. Following ischemia and reperfusion, most B/K-labeled ganglion cells were lost, whereas between 1 day and 2 weeks post-lesion B/K immunoreactivity appeared in many more Muller cell processes with increasing intensity. Quantitative evaluation by immunoblotting confirmed that B/K expression then decreased progressively, to 35% of control values at four weeks post-lesion. Our findings suggest that Muller cells are involved in the pathophysiology of retinal ischemia through the expression of B/K following transient ischemia.

Animals↗

Characterization of the 5'-flanking region of the gene encoding the 50 kDa subunit of human DNA polymerase delta.

DNA polymerase delta consists of at least four subunits: p125, p68, p50, and p12 [Liu et al., J. Biol. Chem. 275 (2000) 18739-18744]. We have isolated genomic DNA clones covering the gene for the human DNA polymerase delta 50 kDa subunit (POLD2) and its 5'-flanking sequence. The POLD2 gene is composed of 11 exons and is distributed over 10 kb of genomic DNA. All exon-intron splice junctions conformed to the GT/AG consensus sequence. The 5'-flanking region of human POLD2 is G+C-rich and does not have a typical TATA box. A computer-based search for potential transcription factor binding sites revealed the existence of a number of motifs including those for AP1, AP2, Sp1, NF-1 and CREB. The functional activity of the regulatory region of the human POLD2 gene was demonstrated by its ability to drive the expression of a chloramphenicol acetyltransferase reporter gene in COS-7 cells.

Animals↗

Identification of phosphorylated form of cAMP/calcium response element binding protein expression in the brain stem nuclei at early stage of vestibular compensation in rats.

The present study was aimed at evaluating the time-dependent expressions of phosphorylated forms in the cAMP/calcium response element binding protein (pCREB) known to be one of transcription factors for immediate early genes in the brain stem nuclei of Sprague-Dalwey rats. Animals received surgical ablation of right peripheral vestibular labyrinth through a ventral approach. Immunohistochemical staining and digital image analysis systems were used to observe pCREB expressions in neuronal cells of the brain stem nuclei. The number of pCREB labeling neurons were increased with time, and peaked in the vestibular nuclear complex (VNC) bilaterally 1 h after unilateral labyrinthectomy (UL). In addition, high levels of phosphorylated CREB-like immunoreactivity (pCREB-LI) were also observed in bilateral cerebellum, olivary nuclear complex, and medullary nuclei. There was a rapid reduction of pCREB-LI in contralateral VNC but a slow reduction in the ipsilateral side 2 h after UL, causing an asymmetric number of pCREB labeling neurons between bilateral VNC (P<0.05). Thereafter pCREB-LI in bilateral VNC decreased to a very low level and returned to basal level 24 h after UL. These results suggest that the activation of the cAMP/calcium response element binding protein plays a role in the initial events of vestibular compensation in rats.

Animals↗

Regulation of protein function by native metastability.

In common globular proteins, the native form is in its most stable state. In contrast, each native form exists in a metastable state in inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins. Metastability in these proteins is critical to their biological functions. Mutational analyses and structural examination have previously revealed unusual interactions, such as side-chain overpacking, buried polar groups, and cavities as the structural basis of the native metastability. However, the mechanism by which these structural defects regulate protein functions has not been elucidated. We report here characterization of cavity-filling mutations of alpha(1)-antitrypsin, a prototype serpin. Conformational stability of the molecule increased linearly with the van der Waals volume of the side chains. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. These results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions.

Amino Acids↗

Upregulation of ciliary neurotrophic factor in reactive Müller cells in the rat retina following optic nerve transection.

We have investigated the expression and cellular localization of ciliary neurotrophic factor (CNTF) in the rat retina following optic nerve transection. In the normal retina, CNTF immunoreactivity was restricted to profiles in the ganglion cell layer. Following optic nerve transection, immunoreactivity appeared in Müller cell somata and processes and its intensity increased between three and seven days post-lesion. Quantitative evaluation by immunoblotting confirmed that CNTF expression increased continuously up to 7 days after optic nerve transection (to 430% of control levels), but decreased again to 250% of controls at 4 weeks post-lesion. Our findings suggest that CNTF supplied by Müller cells may play a protective role for axotomized ganglion cells in the rat retina.

Animals↗

Identification of a fourth subunit of mammalian DNA polymerase delta.

A 12-kDa and two 25-kDa polypeptides were isolated with highly purified calf thymus DNA polymerase delta by conventional chromatography. A 16-mer peptide sequence was obtained from the 12-kDa polypeptide which matched a new open reading frame from a human EST () encoding a hypothetical protein of unknown function. The protein was designated as p12. Human EST was identified as the putative human homologue of Schizosaccharomyces pombe Cdm1 by a tBlastn search of the EST data base using S. pombe Cdm1. The open reading frame of human EST encoded a polypeptide of 107 amino acids with a predicted molecular mass of 12.4 kDa, consistent with the experimental findings. p12 is 25% identical to S pombe Cdm1. Both of the 25-kDa polypeptide sequences matched the hypothetical KIAA0039 protein sequence, recently identified as the third subunit of pol delta. Western blotting of immunoaffinity purified calf thymus pol delta revealed the presence of p125, p50, p68 (the KIAA0039 product), and p12. With the identification of p12 mammalian pol delta can now be shown to consist of four subunits. These studies pave the way for more detailed analysis of the possible functions of the mammalian subunits of pol delta.

Amino Acid Sequence↗

Evidence that DNA polymerase delta isolated by immunoaffinity chromatography exhibits high-molecular weight characteristics and is associated with the KIAA0039 protein and RPA.

DNA polymerase delta, the key enzyme for eukaryotic chromosomal replication, has been well characterized as consisting of a core enzyme of a 125 kDa catalytic subunit and a smaller 50 kDa subunit. However, less is known about the other proteins that may comprise additional subunits or participate in the macromolecular protein complex that is involved in chromosomal DNA replication. In this study, the properties of calf thymus pol delta preparations isolated by immunoaffinity chromatography were investigated. It is demonstrated for the first time using highly purified preparations that the pol delta heterodimer is associated with other polypeptides in high-molecular weight species that range from 260000 to >500000 in size, as determined by FPLC gel filtration. These preparations are associated with polypeptides of ca. 68-70, 34, 32, and 25 kDa. Similar findings were revealed with glycerol gradient ultracentrifugation. The p68 polypeptide was shown to be a PCNA binding protein by overlay methods with biotinylated PCNA. Protein sequencing of the p68, p34, and p25 polypeptide bands revealed sequences that correspond to the hypothetical protein KIAA0039. KIAA0039 displays a small but significant degree of homology to Schizosaccharomyces pombe Cdc27, which, like Saccharomyces cerevisiae Pol32p, has been described as the third subunit of yeast pol delta. These studies provide evidence that p68 is a subunit of pol delta. In addition, the p68-70 and p32 polypeptides were found to be derived from the 70 and 32 kDa subunits of RPA, respectively.

Amino Acid Sequence↗

Inhibitory action of thimerosal, a sulfhydryl oxidant, on sodium channels in rat sensory neurons.

The effects of thimerosal, a sulfhydryl oxidizing agent, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion neurons were studied using the whole-cell patch clamp technique. Thimerosal blocked the two types of sodium channels in a dose-dependent manner. The inhibitory effect of thimerosal was much more pronounced in TTX-R sodium channels than TTX-S sodium channels. The effect of thimerosal was irreversible upon wash-out with thimerosal-free external solution. However, dithiothreitol, a reducing agent, partially reversed it. Thimerosal shifted the steady-state inactivation curves for both types of sodium channels in the hyperpolarizing direction. The voltage dependence of activation of both types of sodium channels was shifted in the depolarizing direction by thimerosal. The inactivation rate in both types of sodium channels increased after thimerosal treatment. All these effects of thimerosal would add up to cause a depression of sodium channel function leading to a diminished neuronal excitability.

Animals↗

Gene expression of flap endonuclease-1 during cell proliferation and differentiation.

It has been shown that flap endonuclease-1 (FEN-1), a structure-specific nuclease, acts on the removal of RNA primers during Okazaki fragment maturation in DNA synthesis. To study whether the gene expression of FEN-1 is inducible during cell proliferation, we analyzed the FEN-1 mRNA levels in actively growing cells and non-growing cells. The gene expression of FEN-1 was higher in mitotic cells than in resting cells, and was markedly decreased, especially, when terminal differentiation was induced in promyelocytic leukemia cells (HL-60 cells). The decline correlated substantially with the ceasing of DNA synthesis. In the examination of tissue-specific gene expression, the human testis, spleen, thymus and mucosal lining of colon tissues expressed this gene actively, whereas the prostate, ovary, small intestine and peripheral blood leukocyte hardly expressed it. In addition, FEN-1 was co-localized with the proliferating cell nuclear antigen (PCNA) in young rat kidney according to immunohistochemistry. These findings suggest that FEN-1 gene expression is inducible during cell proliferation for DNA synthesis, and is down-regulated during cell differentiation.

Animals↗

Differential regulation of ciliary neurotrophic factor and its receptor in the rat hippocampus following transient global ischemia.

To investigate a potential role of ciliary neurotrophic factor (CNTF) in transient global ischemia, we have studied the postischemic regulatory changes in the expression of CNTF and its receptor, the ligand-binding alpha-subunit (CNTFRalpha). Immunoblot analysis demonstrated CNTF levels were slightly upregulated already during the first day after ischemia and then increased markedly by more than 10-fold until 2 weeks postischemia. Immunoreactivity for CNTF became detectable 1 day after ischemia and was localized in reactive astrocytes. The intensity of the immunolabeling was maximal in CA1 during the phase of neuronal cell death (days 3-7 postischemia) and in the deafferented inner molecular layer of the dentate gyrus. Upregulation of CNTF expression was less pronounced in CA3 and absent in the stratum lacunosum moleculare and the outer molecular layer of the dentate gyrus and thus did not simply correlate with astroliosis as represented by upregulation of glial fibrillary acidic protein (GFAP). As shown by in situ hybridization, expression of CNTFRalpha mRNA was restricted to neurons of the pyramidal cell and granule cell layers in control animals. Following ischemia, reactive astrocytes, identified by double labeling with antibodies to GFAP, transiently expressed CNTFRalpha mRNA with a maximum around postischemic day 3. This astrocytic response was most pronounced in CA1 and in the hilar part of CA3. These results show that CNTF and its receptor are differentially regulated in activated astrocytes of the postischemic hippocampus, indicating that they are involved in the regulation of astrocytic responses and the neuronal reorganizations occurring after an ischemic insult.

Animals↗

Increased expression of ciliary neurotrophic factor receptor alpha mRNA in the ischemic rat retina.

Using in situ hybridization, we investigated the expression of ciliary neurotrophic factor receptor ((CNTFRalpha) mRNA in the rat retina rendered ischemic by elevation of the intraocular pressure (IOP). The IOP was increased to 120 mmHg and maintained for 60 min. The rats were sacrificed on the day of reperfusion (DRP) 1, 3, 7, 14, and 28. In the normal retina, the signal for CNTFRalpha mRNA was present in retinal cells in the inner nuclear layer (INL) and in the ganglion cell layer (GCL). On DRP 1, numerous cells in the INL and GCL showed a CNTFRalpha mRNA signal. From DRP 3 onwards, CNTFRalpha mRNA appeared in photoreceptor cells located in the outer part of the outer nuclear layer. The signal in these cells increased up to DRP 14 and then decreased at DRP 28. Our findings suggest that cells expressing CNTFRalpha mRNA may resist the degenerative processes induced by ischemic insult in the rat retina.

Animals↗

The expression and cellular localization of brain/kidney protein in the rat retina.

Brain/kidney (B/K) protein is a new protein of 474 amino acids, which contains two C2 domains structurally homologous to those present in synaptotagmins. The expression of B/K protein was identified in the rat retina and B/K protein immunoreactivity was localized to a number of ganglion cells, a few amacrine cells and the radial processes of Müller cells. Thus, B/K protein appears to be important in the homeostasis in these cells of the rat retina.

Animals↗

Ganglion cells of the rat retina show osteopontin-like immunoreactivity.

Osteopontin (OPN) is a negatively charged, highly acidic glycosylated phosphoprotein that contains an GRGDS amino acid sequence, characteristic of proteins that bind to integrin receptors, thereby playing crucial roles in a number of physiological processes. This study was conducted to examine the expression of OPN in the rat retina by Northern blot analysis, Western blot analysis and immunocytochemistry. The expression of OPN was identified in the retina and OPN-like immunoreactivity was present in a number of ganglion cells. Thus, OPN appears to be important in the retinal homeostasis.

Animals↗