Intracellular Remodeling and Accumulation of Aberrant Lysosomes in Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Parathyroid-Like Cells

Differentiation of mesenchymal stem cells (MSC) into a variety of cell lineages such as adipocytes, osteocytes, and chondrocytes is often accompanied up-regulation of autophagy. In our study, we demonstrated that the expression of autophagy-associated proteins (p-Beclin 1, LC3A, LC3B, p-AMPK, p-mTOR and ATG3, ATG7, and ATG12-5) over a period of time was hardly distinguishable from control tonsil-derived MSC (TMSC). Despite the unnoticeable difference in autophagy activation between differentiated TMSC (dTMSC) and the control (cTMSC), we reported significant changes in intracellular compositions in differentiated TMSC into functional parathyroid-like cells secreting parathyroid hormone (PTH). By using transmission electron microscopy (TEM), we observed accumulation of multivesicular bodies (MVB) comprising small, degraded compartments densely accumulated as dark granular or amorphous clumps, multilamellar bodies and lipid droplets in dTMSC. However, no such structures were found in cTMSC. These results suggest that differentiation of TMSC into parathyroid-like cells producing PTH hormone is hardly dependent on autophagy activation in the beginning of our conditions. Furthermore, our results of intracellular remodeling and accumulated endo-lysosomal storage bodies in the later stages of TMSC differentiation present a possible role of the structures in PTH secretion.

Charged MVB protein 5 is involved in T-cell receptor signaling

Charged multivesicular body protein 5 (CHMP5) has a key role in multivesicular body biogenesis and a critical role in the downregulation of signaling pathways through receptor degradation. However, the role of CHMP5 in T-cell receptor (TCR)-mediated signaling has not been previously investigated. In this study, we utilized a short hairpin RNA-based RNA interference approach to investigate the functional role of CHMP5. Upon TCR stimulation, CHMP5-knockdown (CHMP5(KD)) Jurkat T cells exhibited activation of TCR downstream signaling molecules, such as PKCθ and IKKαβ, and resulted in the activation of nuclear factor-κB and the marked upregulation of TCR-induced gene expression. Moreover, we found that activator protein-1 and nuclear factor of activated T-cells transcriptional factors were markedly activated in CHMP5(KD) Jurkat cells in response to TCR stimulation, which led to a significant increase in interleukin-2 secretion. Biochemical studies revealed that CHMP5 endogenously forms high-molecular-weight complexes, including TCR molecules, and specifically interacts with TCRβ. Interestingly, flow cytometry analysis also revealed that CHMP5(KD) Jurkat T cells exhibit upregulation of TCR expression on the cell surface compared with control Jurkat T cells. Taken together, these findings demonstrated that CHMP5 might be involved in the homeostatic regulation of TCR on the cell surface, presumably through TCR recycling or degradation. Thus CHMP5 is implicated in TCR-mediated signaling.

Detection of microvesicle miRNA expression in ALL subtypes and analysis of their functional roles / 华中科技大学学报（医学）（英德文版）

Microvesicles (MVs) are the heterogeneous mixtures of vesicles. MVs released by leukemia cells constitute an important part of the leukemia microenvironment. MVs might act as important reservoirs of microRNAs (miRNAs). It is worth evaluating whether MVs possess some unique miRNA contents that are valuable in understanding the pathogenesis. In this study, we investigated the miRNA expression patterns of Nalm-6-derived MVs, Jurkat-derived MVs and normal cell-derived MVs using miRNA microarrays. The potential target genes regulated by differentially expressed miRNAs were also predicted and analyzed. Results demonstrated that 182 miRNAs and 166 miRNAs were differentially expressed in Nalm-6-MVs and Jurkat-MVs, respectively. Many oncogenes, tumor suppressors and signal pathway genes were targeted by these aberrantly expressed miRNAs, which might contribute to the development of B-ALL or T-ALL. Our findings expanded the potential diagnostic markers of ALL and provided useful information for ALL pathogenesis.

Ultrastructural Alterations Induced by 5-Fluorouracil or Mitomycin C on the Gastric Parietal Cells of Mouse / 대한해부학회지

This experiment was performed to evaluate the morphological responses of 5-fluorouracil or mitomycin C on the gastric parietal cells of mouse. 5 -fluorouracil (30 mg/kg) or mitomycin C (400 micro gram/kg) were injected subcutaneously every other day, and the animals were sacrificed at 4th day and 7th day following the first injection. Pieces of the tissue were taken from the stomach, prefixed with 2.5% glutaraldehyde -1.5% paraformaldehyde, followed by post-fixation with 1% osmium tetroxide. The ultrathin sections were stained with uranyl acetate and lead citrate. In both of the 5-fluorouracil or the mitomycin C treated groups, most parietal cells showed severely reduced luminal spaces of the intracellular canaliculi, since microvilli of intracellular canaliculi were very irregular shaped and nearly contacted with each other, and the cytoplasmic tubulovesicular membranes were disintegrated and indistinct. The changes in the 5-fluorouracil treated group were more indistinct than in those of the mitomycin C treated group. In the 5-fluorouracil treated group, balooning of the cytoplasm, focal cytolysis, myelin figures, lysosomes and multivesicular bodies in the parietal cells were observed more frequently than in those of the mitomycin C treated group. Above results suggest that the 5-fluorouracil or mitomycin C treated animals might suffer from reduced acid secretion of the parietal cell, since the collapsed lumen of the intracellular canaliculi, the disintegration of the tubulovesicular membranes, and the reduction of cell organelles in the parietal cells are occurred within a few days following injections. 5-fluorouracil was proved more harmful on the parietal cell than mitomycin C does.

Effects of BCG or AG60 on Gastric Parietal Cells of the Mouse Implanted with Ehrlich Carcinoma Cells / 대한해부학회지

This experiment was performed to evaluate the morphological responses of the gastric parietal cells of mouse inoculated with Ehrlich carcinoma cells, following administration of Bacillus Calmette -Guerin (BCG) or acriflavine -guanosine composition (AG60, Taerim Pharm. Co. Seoul, Korea). In the experimental groups, each mouse was inoculated with 1 X 10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. From next day, 0.2 ml of saline, BCG (0.03 X 10(8) ~0.32 X 10(8) CFU) or AG60 (30 mg/kg) was injected subcutaneously to the animals every other day. Animals were sacrificed on the 14th day from the first injection. Pieces of the tissue were taken from the stomach, prefixed with 2.5% glutaraldehyde -1.5% paraformaldehyde, followed by post -fixation with 1% osmium tetroxide. The ultrathin sections stained with uranyl acetate and lead citrate were observed with a JEM 100CX -II electron microscope. In the experimental control, the BCG and the AG60 treated groups, most parietal cells showed reduced lumenal spaces of the intracellular canaliculi, since microvilli of intracellular canaliculi were very irregularly shaped and crowed with each other. And in the BCG and the AG60 treated mice, myelin figures, lysosomes and multivesicular bodies in the parietal cells were observed more frequently than in those of the experimental control ones. In the BCG treated rats, membranes of the tubulovesicles of the parietal cells were disintegrated, but the similar changes were not observed in the AG60 treated mice,. Above results suggest that the BCG treated animals inoculated with Ehrlich carcinoma cells might suffer from reduced acid secretion of the parietal cell, since the disintegration of the tubulovesicular membranes in the parietal cells are occurred following injections. Whereas AG60 dose not affect remakably defect on the parietal cells.

Electron Microscopic Studies on the Effects of Retinoic Acid on Palatogenesis in Fetal Rats / 체질인류학회지

Retinoic acid (RA) is widely used to treat the dermatologic disorders, such as acne and psoriasis, but its usage is limited because of teratogenic effects. Moreover, it is known that RA induces cleft palate by influencing epithelial differentiation and mesenchymal cells in palatine processes. We studied the ultrastructures of the epithelial and mesenchymal cells in rat palatine shelves treated with RA, in comparison with those of the normal developing rat. In this experiment, pregnant Sprague -Dawley rats were treated with 100 mg/kg of all -trans retinoic acid at day 10 of gestation. Pregnant rats were killed at 14 th and 16 th day of gestation. Fetuses were removed and palatine processes were dissected. The specimen were observed with a transmissiom electron microscope. The results were as follows. 1. Palatine epithelium of control rats was made up of two cell layers at day 14 of gestation, and that of RA treated rats consisted of multicellular layers. At the 16th day of gestation, many apoptotic bodies were observed in triangular area of the palatine epithelium of the control rat. In contrast, apoptotic cells were hardly observed in RA treated rats. 2. Mesenchymal cells of control rats contained cytoplasmic process, oval -shaped nucleus, well -developed rough endoplasmic reticulum, Golgi complex, and mitochondria. RA treated mesenchymal cells showed atrophied cisternae of Golgi complex, rough endoplasmic reticulum with sacculated, fragmented and ribosome detached cisternae, mitochondria with dissolved mitochondrial cristae, and multivesicular body. After RA exposure during palatogenesis, the frequency of apoptotic bodies was low in palatine epithelium, and mesenchymal cells were severely damaged. In conclusion, it is suggested the RA may induce direct cytotoxic effects on mesenchymal cells and influence normal apoptosis process in developing epithelium.

Studies on the Seasonal Changes in Epithelium of the Seminiferous Tubules and Serum Concentration of Testosterone , LH and FSH in the Korean Pheasant Phasianus colchicus / 대한해부학회지

It is highly desirable to achieve optimal reproductive performance, reliable morphological and physiological basic data of the reproductive organs. Therefore, seasonal changes in serum testosterone, LH, and FSH concentrations, and morphological changes in testicular epithelial cells were studied in the Korean native pheasant throughout the annual cycle. Mature male pheasants[14-16 months after hatching] were used in this study. The general morphological changes of the epithelia of the seminiferous tubules were observed by dibasic stain, and semithin section from Epon blocks with a phase contrast microscopy. The ultrastructural changes of the the epithelia of the seminiferous tubules were investigated by ultrathin section with transmission electron microscope. The changes in the profiles of the serum FSH, LH, and testosterone concentratioins were measured by RIA[radioimmunoassay]. The results obtained are summarized as follows : 1. There was little variation in the average diameter of the seminiferous tubules from autumn[67.13+/-5.95micrometer] to winter[68.59+/-6.07micrometer], but the highest levels were reached in spring[192.78+/-41.58micrometer]. Thereafter, the diameter decreased slowly in summer[146.57+/-43.68micrometer], then decreased significantly in autumn[67.13+/-5.95micrometer]. 2. Serum testosterne concentration was low from autumn[13.+/-7.21ng/100ml] to winter[17.39+/-13.75ng/100ml], but the highest levels were reached in spring[127.72+/-66.47 ng/100ml]. Thereafter, the concentration was lowest in autumn[13.+/-7.21ng/100ml]. 3. Serum LH concentration increased slowly and linealy from autumn[5.04+/-1.04ng/100ml] to winter[6.23+/-1.08ng/100ml], but the highest levels were reached in spring[11.3+/-3.6 ng/100ml]. Thereafter, the concentration reached the lowest level in autumn[5.04+/-1.04 ng/100ml]. 4. Serum FSH concentration was low from autumn[4.65+/-0.63ng/100ml] to winter[4.2+/-0.98ng/100ml], but the highest levels were reached in spring[17.41+/-8.35ng/100ml]. Thereafter, concentration was the lowest in autumn[4.65+/-0.63ng/100ml]. 5. The seminiferous tubules showed the onset of the spermatongenic cycle in spring but the seminiferous tubules collected in summer exhibited partially degenerative changes. 6. The cytoplasmic process of Sertoli cells of the seminiferous tubules of the pheasant were collected in summer. Many vesicles and degeneratiye changes were included but many number of spermatozoa were embedded partially in the multivesicular bodies in these processes. 7. The diameter of the seminiferous tubules of the pheasant narrowed markedly in autumn, and atrophied in winter. The spermatogonia and Sertoli cells were arranged in monolayer. 8. The myelin figures in the cytoplasmic process of Sertoli cells of the seminiferous tubules of the pheasant in autumn. The nucleus of the Sertoli cells were of a round configuration elongated and oriented perpendicularly to the basement membrane. The results obtained provide basic data for reproductive physiology and are useful for studying the male genital organs of the Korean native pheasant. Structural changes of the seminiferous epithelial cells significantly and postively correlated with serum FSH, LH. The correlation of changes in the hormonal status with alterations of Sertoli cell organells precedes the breeding season.

Two cases of clear cell syringoma / 대한피부과학회지

Two cases of clear cell syringoma are reported in the women who were 39 ad 54 years old, respectively. They had had asymptomatic, ricegrain-sized skin colored to yellowish pink papules on the face, particularly on he lower eyelid for several years. Interestingly, both of them had suffered from diabetes mellitus for more than a decade and been placed on irregular antidiabetic medications. Routine laboratory findings in the elder patient(case 2), unfortunately not checked in the younger patient(cases 1) due to her refusal, were within normal limits except marked glucosuria. Histologic examination revealed numerous various-sized, well-defined tumor nests composed largely of clear cells with polygonal cellular boundary and eccentric nucleus. Special stain with Periodic acid-Schiff disclosed existance of glycogen in the clear cells, but other special stains such as alcian blue showed negative findings. Electron microscopic examination of the elder patient revealed many multivesicular bodies suggestive of lysosomes, and numerous droplets of glycogen dispersed between them in villi-rich periluminal cell.

Ultrastructural Study on the Development of The Aorticopulmonary Body in Human Fetuses

The development of aorticpulmonary bodies was studied by electron microscope in human fatuses ranging from 40mm to 260mm crowm-rump length. The aorticpulmonary bodies were observed in the wall of the aorta, and of the pulmonart trunk and arteries. At 40mm fetus, the aorticopulmonary bodies were composed of clusters of primitive glomus cells, primative supporting cells, unmyelinated nerve fibers, and capillaries. The primitive glomus cells possessed large nuclei, dense-cored vesicles, many Golgi complexes, rough endoplasmic reticulum, and, multivesicular bodies, the primitive supporting cells were agranular with attenuated cytoplasmic processed which partially ensheathed the primitive glomus cells. Synaptic contacts between the axon terminals and the aoma of primitive glomus cells were first observed. The primitive glomus cells increased somewhat in size and number by 90mm fetus, but retained essentially the same characteristics as at the earlier stage. Desmosome-like contacts between glomus cells and adjacent cells were commonly seen. At 160mm fetus, the glomus cells had increased accumulations of all organells and numerous dense cored vesicles. The supporting cells completely invested the glomus cells. Two types of nerve terminals were observed. One type contained small agranular vesicles which was identified as cholinergic axon terminal. The other contained a majority of small granular vesicles which was classfied as adrenergic axon terminal. Synaptic contacts between the cholinergic axon terminals and the soma of the glomus cell were observed. During next prenatal stage up to 260mm fetus the glomus cells and the supporting cells resembling those in adult were present. It is concluded that the ultrastructural features of these aorticopulmonary bodies are similar to those of the carotid body. It is therefore suggested that the aorticopulmonary bodies of the human fetures have a chemorecepter function similar to that of the carotid body.