THE EFFECT OF Ge-132 ON ULTRASTRUCTURE OF CULTURED MELANOCYTES

Objective To elucidate the effect of Ge 132 on the growth of melanocytes.Methods Melanocyes from epidermis were cultured and purified;the second generation of the cell was used for study;the cells were divided into two groups randomly,to group A, Ge 132 was added to the media at 0.04mg/L;to group B,common culturing method was used without Ge 132.After 5d, the cells were seperated by digestion for study by transmission electronic microscope.Results Compared to group B, the vaculoes of the cells were increased,mitochondria distended, endoplasmic reticulum dilated and the number of melanosome declined in the group A.Conclusion Ge 132 can inhibit the melanocytes growth at a certain concentration and might be used for treating pigmented diseases.

Ultrastructure of Amelanotic Melanocytes from Human Hair Follicles

To investigate the ultra structure of amelanotic melanocytes （AMMC）. Methods： The hair follicles obtained from normal human scalp by 0.50% collagenase type V treatment were washed with 0.1 mol/L phosphate buffer salt （PBS）. Hair-follicle cell suspensions were prepared by trypsin treatment and cultured in melanocyte medium. Remaining keratinocytes were removed by differential trypsinization. 100μg/ml geneticin was used to eliminate the contaminating fibroblasts. At third passage, the cells were trypsinized, and then washed in phosphate-buffered saline and processed for transmission electron microscopy. Results： Under transmission electron microscope, the cultured cells showed round or oval shape, with single large nuclear and the karyotheca were double deck. There were obvious euchromosome within the nucleus, and sparse heterochromosome. There were various organelles in the cytoplasm, including plentiful melanosomes with nearly similar size, mitochondria, rough endoplasmic reticule （RER） and ribosome. The electron density granules in most of the melanosomes disposed along concentric circularities. Golgi apparatus in the cells was inconspicuous. Conclusion： The ultra structure of AMMC from human hair follicles is different from that of epidermal melanocytes, and these characteristics determine the functional immature of AMMC.

MicroRNA-370-5p inhibits pigmentation and cell proliferation by downregulating mitogen-activated protein kinase kinase kinase 8 expression in sheep melanocytes

In mammals,microRNAs(miRNAs)play key roles in multiple biological processes by regulating the expression of target genes.Studies have found that the levels of miR-370-5p expression differ significantly in the skins of sheep with different hair colors;however,its function remains unclear.In this study,we investigated the roles of miR-370-5p in sheep melanocytes and found that the overexpression of miR-370-5p significantly inhibited cell proliferation(P<0.01),tyrosinase activity(P=0.001)and significantly reduced(P<0.001)melanin production.Functional prediction revealed that the 3′-untranslated region(UTR)of MAP3K8 has a putative miR-370-5p binding site,and the interaction between these two molecules was confirmed using luciferase reporter assays.In situ hybridization assays revealed that MAP3K8 is expressed in the cytoplasm of melanocytes.The results of quantitative RT-PCR and Western blotting analyses revealed that overexpression of miR-370-5p in melanocytes significantly inhibits(P<0.01)MAP3K8 expression via direct targeting of its 3′UTR.Inhibition of MAP3K8 expression by siRNA-MAP3K8 transfection induced a significant inhibition(P<0.01)of melanocyte proliferation and significant reduction(P<0.001)in melanin production,which is consistent with our observations for miR-370-5p.Target gene rescue experiments indicated that the expression of MAP3K8 in melanocytes co-transfected with miR-370-5p and MAP3K8-cDNA(containing sites for the targeted binding to miR-370-5p)was significantly rescued(P≤0.001),which subsequently promoted significant increases in cell proliferation(P<0.001)and melanin production(P<0.01).Collectively,these findings indicate that miR-370-5p plays a functional role in inhibiting sheep melanocyte proliferation and melanogenesis by downregulating the expression of MAP3K8.

Imiquimod:A potential option for inhibition of repigmentation of congenital melanocytic nevus after laser ablation

Congenital melanocytic nevi(CMN) are common skin tumors. Large and specially located nevi cannot be completely removed by surgery, posing the risks of both cosmetic deformities and potential malignancy.Nonsurgical treatments, such as laser therapy and physical dermabrasion, can overcome the limitations of surgery;however, the high rate of repigmentation remains an unresolved global challenge. We conducted a self-controlled observational study of a patient with a nevus on the chest. Two areas of the lesion were treated with an Er:YAG laser and 5% imiquimod cream was applied to one of these areas. After nearly 7-months of follow-up, we observed a significant difference in color between the two areas, suggesting that topical imiquimod may inhibit repigmentation and significantly enhance the effectiveness of laser treatment.

Physiology and Ultrastructure of Azolla imbricata as Affected by Hg 2+ and Cd 2+ Toxicity

The toxic effects of different gradient concentrations of Hg2+ and Cd2+ on chlorophyll content, chlorophyll a/b value, photosynthetic O-2 evolution, respiration rate, anti-oxidase system (superoxide dismulase (SOD), catalase (CAT), peroxidase (POD)) and ultrastructure of the cells of Azolla imbricata (Roxb.) Nakai were studied. The results showed that with Hg2+ and Cd2+ increase, chlorophyll content and chlorophyll a/b value, photosynthetic O-2 evolution decreased drastically; respiration rate peaked at 2 mg/L pollutant and declined afterwards. The activities of SOD, CAT and POD increased first and decreased afterwards except the activity of POD, which decreased with the increasing of Cd2+ concentration. Ultrastructural observation showed that the extent of ultrastructural damage was much more serious with higher pollutant concentration and longer time of stress. This resulted in swelling of chloroplast, disruption and disappearance of chloroplast membrane and disintegration of chloroplasts; swelling of cristae of mitochondria, deformation and vacuolization of mitochondria; condensation of chromatin in nucleus, dispersion of nucleolus and disruption of nuclear membrane. The experimental results showed: (1) Hg2+ and Cd2+ pollution not only destroyed physiological activities, but also caused irreversible damage to its ultrastructure, thus leading the cells to death; (2) With increase in the stress of Hg2+ and Cd2+, ultrastructural damage was related to the changes of plant physiology; (3) The toxic symptoms of plant showed an evident correlation between dose and effect; (4) The toxicity of Cd2+ on A. imbricata is heavier than that of Hg2+ under the same treatment time and concentration. The lethal concentration of Hg2+ to A. imbricata ranged from 3.5 to 4 mg/L, and that of Cd2+ ranged from 3 to 3.5 mg/L. The damage of cell ultrastructure on Anabaena azollae Strasburger was observed. The results indicated that tolerance of Azolla imbricata for Hg2+ and Cd2+ was higher than that of A. imbricata.

Development and Ultrastructure of the Phloem Ganglion in Bamboo Node

The development, cytological characters and ultrastructure of phloem ganglion in the nodal region of Phyllostachys edulis (Carr.) H. de Lehaie, a most economically important bamboo, were investigated and the possible physiological function of this special structure was proposed. The phloem ganglion derived directly from procambium is situated at the sites where the vascular bundle forks and is present in pairs. The phloem ganglion is spindle_like in appearance and usually consists of 4 to 6 layers. Two kinds of cells in the ganglion could be distinguished. In the middle, there are two layers of filiform cells with pointed ends so that there are no normal sieve plates. Nevertheless, there are many pits on the lateral wall of the filiform cells. The other type of cells located at both ends of the spindle which possess an intermediate form between the filiform cell and the normal sieve tube. The walls of these cells towards the filiform cells are strongly convex forming a special sieve plate. Ultrastructure study showed that cells in the ganglion are connected by enriched plasmodesma. During early differentiation, the paramural body and the ingrowth of cell wall could be observed. It indicates that the cells of phloem ganglion have the character of transfer cells. The organelles in the mature cells are mainly plastids with abundant accumulation of proteins of crystalline structure. The above_mentioned results suggest that the physiological function of the phloem ganglion is closely related with substance transport.

Ultrastructure of Epidermis and Flesh of the Developing Apple Fruit

The ultrastructure of the epidermis and flesh of apple ( Malus domestica Borkh cv. Red Fuji) fruit was systematically observed during the fruit development via transmission electron microscopy. The results showed that, in spite of the ultrastructural changes in many aspects of the developing fruit epidermal cells, it remained almost unchanged throughout the whole developmental process that the cytoplasm was filled with numerous endoplasmic reticula (ER). Most of these endoplasmic reticula were tube_like and rough_ER with enlarged cisterna from which many vesicles were produced. Some of the vesicles were shown to merge into vacuole. Some dynamic Golgi bodies were also found. All the ultrastructural characteristics showed that the epidermal cells have the features of excretory cells. The ultrastructure of the fruit flesh cells at the young fruit stage were shown to be metabolically active, characterized by the presence of numerous clustered plasmodesmata, cisterna enlarged_ and rough_ER filling the cytoplasm, plenty of vesicles and Golgi bodies, indicating their dynamic cellular transport function. Some giant_circular rough_ERs were found. All the ultrastructural features at this early developmental stage should be closely associated with the enlargement of the young fruit. At the rapid growing phase of the fruit the main changes were characterized by: the starch grain_filled amyloplasts, furcating of the single orifice of plasmodesmata, and the cytoplasm enrichment of both the Golgi body_formed vesicles and other vesicles. These features correspond well with those of a photoassimilate sink_cell. An ultrastructural degeneration phenomenon was observed at the fruit ripening stage, but the mitochondria and plasmalemma still remained intact, which might be related to the continuous development of fruit quality during the fruit ripening.

Ultrastructure of the Multicellular Nodules in Hypericum perforatum Leaves

With the development of the multicellular nodules in the leaves of Hypericum per plastids increased both in number and volume, and some plastids degenerated and were surrounded by dark tubular elements and vesicles. Some vesicles fused with vacuoles and secreted substances into vacuoles. There are many multivesicles, multimembrane structures and osmiophilic droplets in vacuoles of nodule cells. Meanwhile, dictyosome secreted vesicles into the vacuoles. However, there is a large central vacuole completely filled with secretory materials (hypericin) in matured multicellular nodules. This suggests that the osmiophilic droplets may be a precursor of hypericin originated from the degenerated plastids. There were abundant endoplasmic reticulums and dictyosomes between the plastids and osmiophilic droplets, suggesting that they may be involved in the synthesis and/or transport of the precursor of hypericin.

Ultrastructure of Oogenesis in Osmunda cinnamomea var. asiatica

The ultrastructure of oogenesis in the fern Osmunda cinnamomea L. var. asiatica Fernald has been studied by electron microscopy. During oogenesis, numerous vesicles not only moved towards the periphery, but also were arranged in line along the inside of plasmalemma, and in addition aggregated outside the plasmalemma by exocytosis. They released or excreted osmiophilic material. It was observed that a few vesicles containing lamellar osmiophilic material situated closely along the plasmalemma, seemed to break open. Simultaneously, a separation cavity between egg cell and archegonium wall formed. Its width was broader than the other advanced ferns reported previously, and an extra egg membrane occurred outside around the plasmalemma of the egg, its thickness being greater than in Pteridium and Dryopteris. Amyloplasts around the nucleus were filled with large triangular semicircular or subelliptical starch grains, but as the egg matured they progressively decreased. Nucleus was large and flattened, and paired membrane in two to three couples laid within the nucleus, close and parallel to the nuclear membrane. No nuclear evagination was observed. Mitochondria seemed to have been undeveloped, but finally recovered normally.

Ultrastructure of the Blepharoplast and the Multilayered Structure in Spermatogenesis in Osmunda cinnamomea var. asiatica

The ultrastructure of the blepharoplast and the multilayered structure (MLS) in the fern Osmunda cinnamomea var. asiatica Fernald have been studied by electron microscopy with respect to spermatogenesis. The blepharoplast appears in the young spermatid. The differentiating blepharoplast is approximately a spherical body, which is composed of densely stained granular material in the center and some cylinders outside of it. The differentiated blepharoplast is also a sphere, but without the densely stained material in the center, consisting of scattered or radially arranged cylinders. The MLS seen in the spermatid lies between the basal bodies and the giant mitochondrion. In the early developmental stage, the MLS only consists of lamellar layers, each of which runs parallel to one another and forms a strip. In the mid stage, the MLS is composed of the microtubular ribbon (MTr), the lamellar layers and a layer of plaque. In the late stage, the MLS forms accessory band, osmiophilic crest and a layer of osmiophilic material. The MTr grows out from the MLS and extends along the surface of the nucleus to unite with the nuclear envelope in a complex. The basal body coming from the cylinder produces the axoneme of the flagella in the distal end and the wedge-shaped structure in the proximal end, respectively. In the present study, the ultrastructural features of blepharoplast and the MLS of the protoleptosporangiopsida fern, O. cinnamomea var. asiatica, have been described and compared with those of other kinds of pteridophytes in detail. The lamellar layers appearing before the formation of the MTr was found and reported for the first time.

ULTRASTRUCTURE OF THE VEGETATIVE CELLS OF NOSTOC FLAGELLIFORME PREPARED WITH HIGH PRESSURE FREEZING AND FREEZE SUBSTITUTION TECHNIQUE

The ultrastructure of the vegetative cells of Nostoc flagelliforme Born. et Flah. was investigated with high pressure freezing and freeze substitution technique and compared with the results obtained by using conventional preparation methods. During the processes of chemical fixation, dehydration and embedding, the cell structures might be more artificially modified than that obtained from high pressure freezing and freeze substitution. With the present method, the sheath of N. flagelliforme could be well penetrated and no extra big space could exist between the cell and the sheath. The cell protoplasm rarely shrinked. Some fine structures of cell inclusions and unit membranes became visualized. Many bacteria were harbored in the sheath. In addition, the presence of big vacuoles in the cell of N. flagelliforme as well as the presence of bacteria in the sheath shown in the present preparation for cyanobacteria has not been described so far in the literature.

Effects of CO_2 Enrichment on Microstructure and Ultrastructure of Two Species of Freshwater Green Algae

order to investigate the morphological response of freshwater green algae to elevated CO2 concentration, Chlamydomonas reinhardtii Dang and Scenedesmus obliquus Kutz were cultured with enriched CO2, and their microstructure and ultrastructure were examined by microscopy and electron microscopy. The effect of CO2 enrichment to 186 mumol/L, was insignificant on the shape and size of C. reinhardtii, but significant in reducing the volume of S. obliquus. High-CO2 increased the amount of chloroplast. The pyrenoids occurred in low-CO2-grown cells but not in high-CO2-grown ones and more starch granules were observed in the former.

Evidence of Ultrastructure and Physiology of F-actin as Component of Plasmodesmata

The characters and ultrastructure of the intercellular connection were revealed in the outer epidermis of the garlic clove sheath by means of fluorescent probe TRITC_Phalloidin (TRITC_Ph) labeling combined with confocal laser scanning microscopy (CLSM), immuno_gold labeling and transmission electron microscopy. These results show that transcellular channel is a complex of rod_like cytoplasm channel and grouped plasmodesmata (PDs) in pit. The former remains a portion of the cell protoplast. The diameter of PD is normally 60-70 nm. The PDs are the real intercellular symplasmic connections of the cells. The transcellular fibers labeled with the TRITC_Ph obviously become narrow in the primary pit fields, which is the same as the characters observed under the electron microscope. The bright fluorescent spot in the primary wall reflects the grouped PDs in pit, and hence the presence of F_actin in the PDs can be confirmed. In immuno_gold labeling experiment, a lot of gold particles were massively distributed in the rod_like cytoplasm channel and grouped PDs. The result provides effective support that these fluorescent filaments possibly are the existing form of F_actin.

THE SECRETORY STIGMAS IN POPULUS: DEVELOPMENT, CYTOCHEMISTRY, ULTRASTRUCTURE AND THEIR RELATION TO INTERSECTIONAL INCOMPATIBILITY *

The stigmas of five species, Populus euphratica Oliv., P. alba L., P. simonii Carr., P. lasiocarpa Oliv. and P. nigra L. have been studied. Scanning electron microscopy re veals that exudates are present in the intercellular spaces, in the clefts between the multicel lular papillae and on the receptive surface. Release and movement of exudates can be visual ized when the fresh stigmas are stained with sudan Ⅲ and auramine O. Paraffin and semi thin resin sections of stigmas after glutaraldehyde osmium fixation evidence the lipidic nature of the exudates. Transmission electron microscopy reveals the glandular features of the stigmatic papillae cells, such as abundance of rough and smooth endoplasmic reticulum, polyribosomes, and well developed dictyosomes with secretory vesicles. Pellicle and epicuticular lamellate layers which have been considered as typical features of the dry type stigmas are also present in the species where stigmas appear extremely wet. It is concluded that stigmas in all of the five species are secretory at the receptive stage. Well developed generative and sperm cells were observed in the pollen tubes penetrating through the deep layers of the stigmatic tissue in the reciprocal crosses between P. euphratica and P. simonii, which indicated that there is no significant barrier in the stigma.

Seasonal Changes in the Ultrastructure of the Vascular Cambium in Shoots of Populus tomentosa

Wood is the product of cambial activity in trees, and the seasonal activity style of cambium directly influences wood biomass production, structures and properties. The seasonal changes in the ultrastructure of the vascular cambium activity of Populus tonientosa Carr. planted in Beijing area were examined in shoot tissues collected during 15 months by means of transmission electron microscopy. Before xylem mother cells reactivated completely, the dividing fusiform cells in cambium and new phloem cells had appeared at the same time. The initiation of cambial activity may be related to the bud sprouting and the young leaf growth in shoots. More details about the ultrastructural changes of cambial cells at the onset of cambial activity have been gained. When the large vacuole in active cambial cells divided into smaller ones during the dormant phase, proteinaceous material that disappeared in active cambial cells refilled many of these small vactioles. In addition, lipid droplets and starch granules had the same cycles as proteinaceous material. The plasmalemma invaginations of fusiform cells were observed not only in active phase but also in dormancy. The endomembrane system consisting of nuclear membrane, endoplasmic reticulum (ER), dictyosomes and their secretory vesicles, changed in form and distribution at different phases during a cycle and performed important roles at the onset of active cambium and during the wall formation process of secondary xylem cells. The tangential walls remained relatively thin throughout the year but the radial walls thickened markedly when the cambium was dormant. During the transition from dormancy to activity, a partial autolysis occurred in the radial walls of the cambial cells, especially at the cell wall junctions. A notable feature of the cells at the onset of cambial activity was the thinning of the radial walls.

Effects of Doubled_CO_2 Concentration on Ultrastructure, Supramolecular Architecture and Spectral Characteristics of Chloroplasts from Wheat

Wheat ( Triticum aestivum L.) plants were grown under ambient and doubled_CO 2(plus 350 μL/L) concentration in cylindrical open_top chamber to examine their effects on the ultrastructure, supramolecular architecture, absorption spectrum and low temperature (77 K) fluorescence emission spectrum of the chloroplasts from wheat leaves. The results were briefly summarized as follows: (1) The wheat leaves possessed normally developed chloroplasts with intact grana and stroma thylakoid membranes; The grana intertwined with stroma thylakoid membranes and increased slightly in stacking degree and the width of granum, in spite of more accumulated starch grains within the chloroplasts than those in control; (2) The particle density in the stacked region of the endoplasmic fracture face (EFs) and protoplasmic fracture face (PFs) and in the unstacked region the endoplasmic fracture face (EFu) and the protoplasmic fracture face (PFu) was significantly higher than that of control. Furthermore, in some cases many more particles on EFs faces of thylakoid membranes appeared as a paracrystalline particle array; (3) The variations in the structure of chloroplasts were consistent with the absorption spectra and the low temperature (77 K) fluorescence emission spectra of the chloroplasts developed under the doubled_CO 2 concentration. Results indicate that the capability of light energy absorption of chloroplasts and regulative capability of excitation energy distribution between PSⅡ and PSⅠ were raised by doubled_CO 2 concentration. This is very favorable for final productivity of wheat.

Obsevation for Epidermal Ultrastructure of Nostoc sphaeroides Kutzing under Scanning Electron Microscope

[Objective]The experiment aimed to explore a new way for observing surface structure of Nostoc sphaeroides Kutzing. [Method] The scanning electron microscope was used to observe the epidermal ultrastructure of wild and cultured Nostoc sphaeroides Kutzing. [ Result] The epidermis of wild and cultured Nostoc sphaeroides Kutzing showed mixture structure of fibril colloid which was reticular arranged. The difference between wild and cultured Nostoc sphaeroides Kutzing was that the outer epidermis of cultured Nostoc sphaeroides Kutzing had trichome distribution but the wild Nostoc sphaeroides Kutzing did not has such distribution. The obsevation results of under smaller than 10 μm by scanning electron microscope was touched thick and showed many folds and distortions. [ Conclusion] The scanning electron microscope was an effective way to study development of Nostoc sphaeroides Kutzing colony and it was worth popularizing.

Effects of Cryopreservation on the Quality and Ultrastructure of Tibetan Mastiff Sperm

[Objective] This study aimed to investigate the effects of cryopreservation on the quality and ultrastructure of Tibetan Mastiff sperm. [Method] The effects of cryopreservation on the quality of Tibetan Mastiff sperm were evaluated via motility, membrane integrity rate and acrosome intact rate. On that basis, the effects of cryopreservation on ultrastructure of sperm were observed under SEM and TEM. [Result] In Experiment 1, EG gave the best results not only in post-thaw motility rate （36.3%）, but also in low membrane integrity rate （38.0%） and acrosome intact rate （42.0% ）, but there was no significant difference between EG group and Glycerol group （P0.05）. In Experiment 2, the 5 cm freezing height obtained the best freezing-thawing results, but there was no significant difference between 2 and 5 cm height （P 0.05）, besides in acrosome intact rate. In Experiment 3, SDS and Vc added separately or together into extenders could improve freezing-thawing results, but there was not obvious difference between SDS group and Vc group （P0.05）, besides the lower motility of Vc group （P0.05）. Addition of SDS and Vc obtained the best results in post-thaw motility rate （44.1%）, and also in membrane integrity rate （48.0%） and acrosome intact rate （48.2%）. The ultrastructure of frozen-thawed sperm was also evaluated under SEM and TEM, results showed that cryopreservation caused various degrees of damage to Tibetan Mastiff sperm, more serious damages were observed in the acrosome such as swelling, vesiculation and even disappearance. [Conclusion] This study confirms that EG, horizontal height of 0.25 ml straw above LN 2 surface and additives SDS and Vc together can improve freezing effect. However, cryopreservation has certain damage to ultrastructure of sperm.

Observation of Ultrastructure in Lesions of Condyloma Acuminatum

Objective: To explore the effect of morphological changes in the development of condyloma acuminatum (CA) Materials and Methods: Lesions in five patients with CA were observed . Results: Upon electron microscopy, the most characteristic feature of the lesions important for diagnosis, was the presence of distinct perinuclear vacuolizations, or so-called koilocytes, among some epithelial cells. These cells possessed hyperchromatic nuclei, swollen mitochondria, dilated endoplasmic reticulum and dissolved glycogen. There were interchromatin granules and perichromatin granules in some nuclei. Moreover some virus particles were also seen in the nuclei of some infected cells. Conclusions: The ultrastructural findings may be used to histopathologically explain the pathogenesis and mechanism of this disease, and it is helpful for diagnosis of CA .

Ultrastructure Features of Sperm Cell in Amaryllis vittata

Ultrastructure of sperm cells in pollen tube of Amaryllis vittata Ait. has been investigated in details by electron microscopy ,with particular emphasis on the organization and distribution of microtubules.The two newly formed sperm cells are arranged in tandem and sometimes in transverse at the right angle to the long axis of the pollen tube.Thevegetative nucleus is ahead and closely associated with the two sperm cells in all examined pollen tubes. The microtubules are distributed in the region between the common cell wall and the proximity of the sperm nucleus,they are singles and dispersed with mainly orientation of longitudinal and oblique,forming a loose bucket-like structure as a whole.In late stage of development,all of the microtubule array longitudinally and enclose the sperm nucleus.This configuration is very similar to the basket-like structure of microtubule in the generative cell. These results show that the organization and distribution of microtubules in the sperm cells are dynamic during cell development.