Ji-Yeon Lee

Differential regulation of osteopontin receptors, CD44 and the αv and β3 integrin subunits, in the rat hippocampus following transient forebrain ischemia

We have examined the spatiotemporal regulation of CD44 and the alpha(v)beta(3) integrin subunits, which have been identified as receptors for osteopontin (OPN), in the rat hippocampus following transient forebrain ischemia. Immunoreactivity for CD44 and the integrin subunits, alpha(v) and beta(3), showed characteristic time- and cell-dependent patterns in the ischemic hippocampus. CD44 immunoreactivity was induced at day 1 after reperfusion, reached a peak at day 3, and returned to basal levels by day 7. CD44 was induced in a subset of activated microglial cells within sites of intense neural damage, and the concomitant induction of OPN and CD44 was observed in the same cells in the ischemic hippocampus. In contrast, increased immunoreactivity for alpha(v) and beta(3), which shared overlapping expression patterns in the ischemic hippocampus, occurred in the majority of reactive astrocytes and only a few microglia at day 3 after reperfusion, and was sustained for more than 2 weeks. Immunoreactivity for both integrin subunits colocalized with OPN immunoreactivity in reactive astrocytes, and OPN immunoreactivity was also diffusely localized over the extracellular matrix around the reactive astrocytes. These data indicated that the rapid and transient induction of CD44 and OPN occurred in activated microglia/macrophages, whereas the long-lasting induction of alpha(v) and beta(3) integrin subunits and OPN occurred in reactive astrocytes, suggesting that the multifunctional role of OPN in the ischemic brain may be attributed, in part, to a time- and cell-dependent interaction with CD44 or integrin alpha(v)beta(3).

Expression of vascular endothelial growth factor receptor-3 mRNA in the rat developing forebrain and retina

Vascular endothelial growth factor receptor (VEGFR)‐3, a receptor for VEGF‐C and VEGF‐D, is expressed in neural progenitor cells, but there has been no comprehensive study of its distribution in the developing brain. Here, the temporal and cell‐specific expression of VEGFR‐3 mRNA was studied in the developing rat forebrain and eye. Expression appeared along the ventricular and subventricular zones of the lateral and third ventricles showing ongoing neurogenesis as early as embryonic day 13 but was progressively down‐regulated during development and remained in the subventricular zone and rostral migratory stream of the adult forebrain. VEGFR‐3 expression was also detectable in some differentiating and postmitotic neurons in the developing cerebral cortex, including Cajal‐Retzius cells, cortical plate neurons, and subplate neurons. Expression in the subplate increased significantly during the early postnatal period but was absent by postnatal day 14. It was also highly expressed in nonneural tissues of the eye during development, including the retinal pigment epithelium, the retinal ciliary margin, and the lens, but persisted in a subset of cells in the pigmented ciliary epithelium of the adult eye. In contrast, there was weak or undetectable expression in the early neural retina, but a subset of retinal neurons in the postnatal and mature retina showed intense signals. These unique spatiotemporal mRNA expression patterns suggest that VEGFR‐3 might mediate the regulation of both neurogenesis and adult neuronal function in the rat forebrain and eye. J. Comp. Neurol. 518:1064–1081, 2010.

Polymorphisms of Cytochrome P450 2D6 are associated with blood hydroxychloroquine levels in patients with systemic lupus erythematosus

To evaluate associations of genetic polymorphisms in cytochrome P450 (CYP) isoforms 2D6, 3A5, and 3A4 with blood concentrations of hydroxychloroquine (HCQ) and its metabolite, N‐desethyl HCQ (DHCQ), in patients with systemic lupus erythematosus (SLE).

Association of Polymorphisms of Cytochrome P450 2D6 With Blood Hydroxychloroquine Levels in Patients With Systemic Lupus Erythematosus

To evaluate associations of genetic polymorphisms in cytochrome P450 (CYP) isoforms 2D6, 3A5, and 3A4 with blood concentrations of hydroxychloroquine (HCQ) and its metabolite, N‐desethyl HCQ (DHCQ), in patients with systemic lupus erythematosus (SLE).

Intravitreal delivery of mesenchymal stem cells loaded onto hydrogel affects the regulatory expression of endogenous NGF and BDNF in ischemic rat retina

The utility of a biodegradable hyaluronic acid (HyA)-based hydrogel as a tissue scaffold for intravitreal carriage of mesenchymal stem cells (MSCs) into the retina was tested. A rat model of retinal ischemia-reperfusion (IR) injury was used. DiI-labeled MSCs from three passages were loaded onto hydrogel and injected into the vitreous body of experimental and control eyeballs 1 week after IR. The neutral hydrogel was also modified to a basic pH. A control carrier, 0.01 M phosphate-buffered saline (pH 7.2; PBS) was compared. Retinal tissues were prepared 1 week and 2 weeks after MSCs application. MSCs localization and effects were evaluated on immunostained retinal preparations with confocal microscopy. MSCs localization was apparent in the retinas loaded onto hydrogels, adhering tightly to the inner limiting membrane, whereas those in PBS floated in the vitreous body. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were expressed in the end feet of Müller cells in the normal retina. NGF expression was slightly reduced 2 weeks IR, had expanded into the proximal processes 3 weeks IR, but it appeared reversely 1 week and 2 weeks after MSCs application, respectively. BDNF expression was higher in the ischemic and MSCs-treated ischemic retinas than in the normal and MSCs-treated control retinas, respectively. These findings demonstrate that HyA-based hydrogel is an efficient vehicle for intravitreal MSC transplantation into the retina, and that MSCs thus transplanted induce Müller glial cells to produce growth factors concerned with the survival of retinal ganglion cells.

Factors related to blood hydroxychloroquine concentration in patients with systemic lupus erythematosus

To identify factors associated with blood concentrations of hydroxychloroquine (HCQ) and its major metabolite, N‐desethylhydroxychloroquine (DHCQ), in patients with systemic lupus erythematosus (SLE; lupus) receiving long‐term oral HCQ treatment.

Estimation of phosphoenolpyruvate carboxylation mediated by phosphoenolpyruvate carboxykinase (PCK) in engineered Escherichia coli having high ATP.

We have previously reported that phosphoenolpyruvate carboxykinase (PCK) overexpression under glycolytic conditions enables Escherichia coli to harbor a high intracellular ATP pool resulting in enhanced recombinant protein synthesis. To estimate how much PCK-mediated phosphoenolpyruvate (PEP) carboxylation is contributed to the ATP increase under engineered conditions, the kinetics of PEP carboxylation by PCK and substrate competing phosphoenolpyruvate carboxylase (PPC) were measured using recombinant enzymes. The PEP carboxylation catalytic efficiency (kcat/Km) of the recombinant PCK was 660mM(-1)min(-1), whereas that of the recombinant PPC was 1500mM(-1)min(-1). Under the presence of known allosteric effectors (fructose 1,6-bisphosphate, acetyl-CoA, ATP, malate, and aspartate) close to in vivo conditions, the catalytic efficiency of PCK-mediated PEP carboxylation (84mM(-1)min(-1)) was 28-folds lower than that of PPC (2370mM(-1)min(-1)). To verify the above results, an E. coli strain expressing native PCK and PPC under control of identical promoter was constructed by replacing PCK promoter region with that of PPC in chromosome. The native PCK activity (33nmol/mg-proteinmin) was 5-folds lower than PPC activity (160nmol/mg-proteinmin) in the cell extract from the promoter-exchanged strain. Intracellular modifications of ATP concentration by PCK activity and the consequences for biotechnology are further discussed.

Transient expression of Bis protein in midline radial glia in developing rat brainstem and spinal cord

Bis (Bcl-2 interacting death suppressor) has been reported to contribute to the differentiation and maturation of specific neuronal populations in the developing rat forebrain, in addition to its well-established functions as a stress or survival-related protein. In the present study, we have analyzed the expression of Bis in the rat brainstem and cervical spinal cord during development by using immunohistochemistry. Bis immunoreactivity was detected in radial glial cells flanking the midline from embryonic day 14. During embryonic and early postnatal development, Bis expression persisted in differentiating radial glia at the midline but disappeared first in the spinal cord by postnatal day 7 (P7) and later also in the brainstem by P14. Bis expression was restricted to a subpopulation of the midline radial glia, i.e., the dorsal midline of the midbrain and spinal cord and the ventral midline of the hindbrain, which were double- or triple-labeled with vimentin and nestin, markers for radial glia, and S100B. However, these markers also labeled all radial glia including the ventral midline glia in the midbrain and spinal cord, with Bis being absent from these structures. In addition, the dorsal midline glia in the midbrain and spinal cord expressed Bis prior to the timing of expression for radial glial markers. Therefore, our results demonstrate the early and transient expression of Bis in the subpopulation of midline glia in the developing brainstem and spinal cord, suggesting that Bis has a unique role in association with the radial glial cells in the developing central nervous system.

Functional Analysis and Immunochemical Analyses of Ca2+ Homeostasis-Related Proteins Expression of Glaucoma-Induced Retinal Degeneration in Rats

The retinal degeneration resulting from elevated intraocular pressure was evaluated through functional and morphological analyses, for better understanding of the pathophysiology of glaucoma. Ocular hypertension was induced via unilateral episcleral venous cauterization in rats. Experimental time was set at 1 and 3 days, and 1, 2, 4, and 8 weeks post-operation. Retinal function was analyzed using electroretinography. For morphological analysis, retinal tissues were processed for immunochemistry by using antibodies against the calcium-sensing receptor and calcium-binding proteins. Apoptosis was analyzed using the TUNEL method and electron microscopy. Amplitudes of a- and b-wave in scotopic and photopic responses were found to be reduced in all glaucomatous retinas. Photopic negative response for ganglion cell function significantly reduced from 1-day and more significantly reduced in 2-week glaucoma. Calcium-sensing receptor immunoreactivity in ganglion cells remarkably reduced at 8 weeks; conversely, protein amounts increased significantly. Calcium-binding proteins immunoreactivity in amacrine cells clearly reduced at 8 weeks, despite of uneven changes in protein amounts. Apoptosis appeared in both photoreceptors and ganglion cells in 8-week glaucomatous retina. Apoptotic feature of photoreceptors was typical, whereas that of ganglion cells was necrotic in nature. These findings suggest that elevated intraocular pressure affects the sensitivity of photoreceptors and retinal ganglion cells, and leads to apoptotic death. The calcium-sensing receptor may be a useful detector for alteration of extracellular calcium levels surrounding the ganglion cells.

Blue-on-Green Flash Induces Maximal Photopic Negative Response and Oscillatory Potential and Serves as a Diagnostic Marker for Glaucoma in Rat Retina

The purpose of this study was to investigate the application of various electroretinography (ERG) to the diagnosis of inner retinal dysfunction induced by mild intraocular pressure (IOP) elevation in a rat glaucoma model. For inner retinal function measurements, available photopic ERG protocols were applied under various light conditions including monochromatic combinations, which complement conventional scotopic ERG. Three episcleral veins in the right eyes of Sprague-Dawley rats were cauterized to induce an experimental model of glaucoma, leading to mild IOP elevation. ERG responses were measured before surgery and at 1, 2, 4, and 8 weeks after cauterization. We first confirmed that the amplitude reduction in the standard photopic b-wave was almost comparable to the amplitudes of scotopic a- and b-waves in glaucomatous eyes over time. We have implemented additional photopic ERG protocols under different stimulus conditions, which consisted of a longer duration and different monochromatic combinations. Such a change in the stimulations resulted in more pronounced differences in response between the two groups. Especially in normal animals, blue stimulation on a green background produced the largest b-wave and photopic negative response (PhNR) amplitudes and caused more pronounced oscillatory potential (OP) wavelets (individual components). In glaucomatous eyes, blue stimulation on a green background significantly reduced PhNR amplitudes and abolished the robust OP components. These results, by providing the usefulness of blue on green combination, suggest the applicable photopic ERG protocol that complements the conventional ERG methods of accessing the progression of glaucomatous damage in the rat retina.