Biomedical subjects

Murat T Budak

Publications and source records attributed to Murat T Budak.

7 recordsLinked to original sources

PubMed2006

Identification of the A3 adenosine receptor in rat retinal ganglion cells.

PURPOSE: Adenosine can protect retinal ganglion cells from the death that accompanies a general ischemic challenge as well as excitotoxic death. In other tissues, both A1 and A3 adenosine receptor subtypes can mediate protection. While a role for the A1 adenosine receptor in ganglion cell protection has been established, a potential for the A3 receptor has only recently been proposed. Although the pharmacology is promising, the molecular identity of the responsible receptor is unclear as previous studies were unable to detect message for the A3 receptor in retinal ganglion cells. We combined laser capture microdisection (LCM) and immunopurification with traditional and real-time PCR to unequivocally demonstrate the presence of the A3 receptor message in rat retinal ganglion cells. METHODS: Retinal ganglion cells of Long-Evans rat pups were retrograde labeled with aminostilbamidine. Eyeballs were enucleated, embedded, frozen, sectioned, and fluorescent cells in the ganglion cell layer were collected with LCM. Purified ganglion cells were also isolated with a two-step panning procedure. cDNA for the A3 receptor obtained from the microdissected ganglion cell layer, immunopurified ganglion cells, whole retina and testis was amplified using RT-PCR, confirmed by DNA sequencing and compared with published sequences. A3 receptor message was also amplified using real-time PCR. Ca2+ levels in immunopanned ganglion cells were measured ratiometrically with fura-2. RESULTS: RNA from immunopurified ganglion cells and from dye-loaded cells in the ganglion cell layer contained message for the A3 receptor when amplified with either traditional RT-PCR or real-time PCR. The entire encoding region was sequenced and found to be 99% identical to the published code. The sequence closely resembled the consensus form of the gene, with other sequences deviating from this default code. Molecular identification was functionally confirmed in purified ganglion cells as the A3 receptor agonist Cl-IB-MECA prevented the excessive Ca2+ rise triggered by P2X7 agonist BzATP. CONCLUSIONS: Retinal ganglion cells express A3 adenosine receptor mRNA. Stimulation of this receptor can reduce the Ca2+ overload following excessive activation of P2X7 receptors.

Adenosine↗

PubMed2005

Definition of the unique human extraocular muscle allotype by expression profiling.

The extraocular muscles (EOMs) are a unique group of specialized muscles that are anatomically and physiologically distinct from other skeletal muscles. Perhaps the most striking characteristic of the EOMs is their differential sensitivity to disease. EOMs are spared in Duchenne's muscular dystrophy (DMD) despite widespread involvement of other skeletal muscles. Conversely, they are early and prominent targets in myasthenia gravis and mitochondrial myopathies. It is unclear how EOMs achieve such specialization or a differential response to diseases; however, this has been attributed to a unique, group-specific pattern of gene expression or "allotype." To begin to address these issues as well as define the human EOM allotype, we analyzed the human EOM transcriptome using oligonucleotide-based expression profiling. Three hundred thirty-eight genes were found to be differentially expressed in EOM compared with quadriceps femoris limb muscle, using a twofold cutoff. Functional characterization revealed expression patterns corresponding to known metabolic and structural properties of EOMs such as expression of EOM-specific myosin heavy chain (MYH13) and high neural, vascular, and mitochondrial content, suggesting that the profiling was sensitive and specific. Genes related to myogenesis, stem cells, and apoptosis were detected at high levels in normal human EOMs, suggesting that efficient and continuous regeneration and/or myogenesis may be a mechanism by which the EOMs remain clinically and pathologically spared in diseases such as DMD. Taken together, this study provides insight into how human EOMs achieve their unique structural, metabolic, and pathophysiological properties.

Adolescent↗

PubMed2005

Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells.

When cell populations are incubated with the DNA-binding dye Hoechst 33342 and subjected to flow cytometry analysis for Hoechst 33342 emissions, active efflux of the dye by the ABCG2/BCRP1 transporter causes certain cells to appear as a segregated cohort, known as a side population (SP). Stem cells from several tissues have been shown to possess the SP phenotype. As the lack of specific surface markers has hindered the isolation and subsequent biochemical characterization of epithelial stem cells this study sought to determine the existence of SP cells and expression of ABCG2 in the epithelia of the ocular surface and evaluate whether such SP cells had features associated with epithelial stem cells. Human and rabbit limbal-corneal and conjunctival epithelial cells were incubated with Hoechst 33342, and analyzed and sorted by flow cytometry. Sorted cells were subjected to several tests to determine whether the isolated SP cells displayed features consistent with the stem cell phenotype. Side populations amounting to <1% of total cells, which were sensitive to the ABCG2-inhibitor fumitremorgin C, were found in the conjunctival and limbal epithelia, but were absent from the stem cell-free corneal epithelium. Immunohistochemistry was used to establish the spatial expression pattern of ABCG2. The antigen was detected in clusters of conjunctival and limbal epithelia basal cells but was not present in the corneal epithelium. SP cells were characterized by extremely low light side scattering and contained a high percentage of cells that: showed slow cycling prior to tissue collection; exhibited an initial delay in proliferation after culturing; and displayed clonogenic capacity and resistance to phorbol-induced differentiation; all features that are consistent with a stem cell phenotype.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2004

Layer-specific differences of gene expression in extraocular muscles identified by laser-capture microscopy.

In mammals, separate muscles are typically specialized as a whole to provide distinct functional roles leading to well-recognized adaptations. This is exemplified in the lower limb by the slow, fatigue-resistant soleus, which provides a postural role vs. the fast, fatiguable tibialis anterior (TA), which provides rapid movements. A unique characteristic of extraocular muscles (EOMs) is their compartmentalization into two distinct layers, the orbital layer (OL) and global layer (GL), presumably to subserve diverse functions within the same muscle. However, molecular evidence of this diversity has been limited. We used laser-capture microscopy coupled with microarray-based expression profiling to identify molecular differences between the OL and GL of rat EOMs. We found that 210 genes were differentially regulated between these layers at a twofold expression cutoff. Differences in genes related to metabolic pathways and related to structural elements of muscle and nerve formed the largest functional clusters. Layer-specific differential expression was validated at both mRNA and protein level for MYH3, MYH6, and ACTN3. The expected layer-specific differences among genes encoding vascular elements were not evident by profiling; morphometric analysis demonstrated that the differences exist, but at a magnitude below the cutoff level established by our statistical methods. Comparison of these results with previous results comparing whole EOMs and TA suggest evolutionary mechanisms may play a role in achieving functional distinctions between OL and GL.

Actinin↗

PubMed2004

Ocular surface epithelial and stem cell development.

Phenotypic features and developmental events involved in the genesis of the limbo-corneal and conjunctival epithelia are described. Together, these two epithelia define the ocular surface. They derive from a small cohort of optic vesicle-induced PAX6+ head ectodermal cells that remain on the surface following lens vesicle formation by the main PAX6+ cell cohort. Both epithelia are stratified, and display wet, non-keratinizing phenotypes. The most significant spatial feature of the limbo-corneal epithelium is the segregation of its supporting stem and early precursor cells to the limbus, the outer vascularized rim separating the cornea from the conjunctiva. These stem cells express ABCG2, a xenobiotic transporter present in stem cells from other organs. ABCG2 transport activity excludes the DNA dye Hoechst 33342, allowing the isolation of the ocular stem cells by flow cytometry, as a unique cohort known as a side 'side population'. Limbal stem cells do not form gap junctions and exist as metabolically isolated entities. Tracking of expression changes in Cx43, the main gap junction protein expressed in both the pre-epithelial ectoderm and in the mature central corneal epithelium, indicates that a limbal stem cell phenotype starts developing very soon after lens vesicle invagination, in advance of the appearance of any recognizable anatomical sub-epithelial limbal feature. Differences in Cx43 expression also reveal the very early nature of the divergence in limbo-corneal and conjunctival lineages. The putative involvement of several early genes, including gradients of PAX6 and differences in expression patterns for members of the Id or msh gene expression regulators are reviewed.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2003

Different cell sizes in human limbal and central corneal basal epithelia measured by confocal microscopy and flow cytometry.

PURPOSE: In the epidermis, the highest clonogenicity, a feature of stem cells (SCs), is found in the smallest keratinocyte. In the limbal-corneal (LC) epithelium the SCs are exclusively localized in the basal epithelial layer of the limbal domain. The current study was conducted to determine whether this spatial SC arrangement is reflected in differences in the cell size between limbal and corneal cells. METHODS: In vivo confocal microscopy was used to scan and measure the size of the cells of the central cornea and the superior limbus in five normal subjects, from the superficial to the basal cell layer. Limbal and corneal pure epithelial sheets were isolated by dispase digestion from human tissues and dissociated into single cells by trypsin digestion. The forward (FSC; a relative measure of cell size) and side (SSC; a relative measure of cytoplasmic complexity) light-scattering properties of these cells were determined by flow cytometry. RESULTS: Confocal microscopy showed that diameters of the basal cells of the limbal and corneal zones were 10.1 +/- 0.8 and 17.1 +/- 0.8 micro m, respectively. The corresponding values for the superficial layers were 19.9 +/- 1.6 and 36.6 +/- 1.6 microm, respectively (P < 0.0001). The mean FSC and SSC of the limbal cells amounted to 65.7% +/- 8.7% and of the corneal cells, 74.4% +/- 4.6%. Furthermore, only 1.40% +/- 0.83% and 0.69% +/- 0.37% of the corneal cells had FSC and SSC equal to the lowest 15% of FSC and SCC of the limbal cells, respectively, indicating that the limbus contained a substantial proportion of very low FSC and SSC cells for which there was no corneal counterpart. CONCLUSIONS: The data collectively demonstrate that the smallest cells are located in the limbal basal epithelium. This feature may help isolate corneal SCs located in the limbus.

Adult↗

PubMed2002

Changes in connexin43 in early ocular surface development.

PURPOSE: In the limbo-corneal epithelium the stem and early precursor epithelial cell pool is confined to the limbal rim. Among the features associated with this spatial segregation is the general paucity of connexin43 (Cx43) within the limbal basal cell population and its complete absence in resident stem cells. The limbo-corneal epithelial lineage derives from a Cx43-positive (Cx43+) embryonic outer ectoderm. Accordingly, as a means of identifying the process through which limbal cell phenotypes emerge, we investigated the expression of Cx43 in the ocular surface of embryonic rats. METHODS: Ocular surface expression of Cx43 or K12 was determined in cryostat sections of rat embryos and eyes using immunohistological methods. RESULTS: Changes in Cx43 expression revealed the early phenotypic divergence of three main epithelial cell phenotypes of the ocular surface. An analysis of the level and distribution pattern of Cx43 puncta lead to the identification of two distinct domains by embryonic day 10 (E10), a stage that occurs soon after formation of the lens vesicle. Additionally, at E12, ectodermal cells directly adjacent to the edges of the developing retina no longer express connexin. A comparison of anatomical and expression changes throughout embryonic development demonstrated that the two early zones represent the rudiments for the epithelia of the central cornea and conjunctiva, respectively, and that the isolated Cx43-negative (Cx43-) cells represent the precursors of the basal and, putatively, stem cells of the limbal epithelium. CONCLUSIONS: Changes in Cx43 expression revealed that the phenotypic divergence of ocular surface epithelial cells and the generation of limbo-corneal stem cell precursors takes place at a very early stage in ocular development, ahead of the establishment of any identifiable anatomical or differentiation features for these domains.

Animals↗