Electron-dense Cytoplasm Research Articles

“CandidatusAnadelfobacter veles” and “CandidatusCyrtobacter comes,” Two NewRickettsialesSpecies Hosted by the Protist CiliateEuplotes harpa(Ciliophora, Spirotrichea)

The order Rickettsiales (Alphaproteobacteria) is a well-known group containing obligate endocellular prokaryotes. The order encompasses three families (Rickettsiaceae, Anaplasmataceae, and Holosporaceae) and a fourth, family-level cluster, which includes only one candidate species, "Candidatus Midichloria mitochondrii," as well as several unnamed bacterial symbionts. The broad host range exhibited by the members of the "Candidatus Midichloria" clade suggests their eventual relevance for a better understanding of the evolution of symbiosis and host specificity of Rickettsiales. In this paper, two new bacteria belonging to the "Candidatus Midichloria" clade, hosted by two different strains of the ciliate protist Euplotes harpa, are described on the basis of ultrastructural observations, comparative 16S rRNA gene sequence analysis, and an estimation of the percentage of infection. Ultrastructure of these bacteria shows some unusual features: one has an electron-dense cytoplasm, and the other one lacks a symbiosomal membrane. The latter was up to now considered an exclusive feature of bacteria belonging to the family Rickettsiaceae. 16S rRNA gene phylogenetic analysis unambiguously places the new bacteria in the "Candidatus Midichloria" clade, although their phylogenetic relationships with other members of the clade are not clearly resolved. This is the first report of a ciliate-borne bacterium belonging to the "Candidatus Midichloria" clade. On the basis of the data obtained, the two bacteria are proposed as two new candidate genera and species, "Candidatus Anadelfobacter veles" and "Candidatus Cyrtobacter comes."

Establishment of a protocol for obtention of neuronal stem cells lineages from the dog olfactory epithelium

A morphological and cell culture study from nasal mucosa of dogs was performed in order to establish a protocol to obtain a cell population committed to neuronal lineage, as a proposal for the treatment of traumatic and degenerative lesions in these animals, so that in the future these results could be applied to the human species. Twelve mongrel dogs of 60-day aged pregnancy were collected from urban pound dogs in São Paulo. Tissue from cribriform ethmoidal lamina of the fetuses was collected at necropsy under sterile conditions around 1h to 2h postmortem by uterine sections and sections from the fetal regions described above. Isolated cells of this tissue were added in DMEM/F-12 medium under standard conditions of incubation (5% CO², >37ºC). Cell culture based on isolated cells from biopsies of the olfactory epithelium showed rapid growth when cultured for 24 hours, showing phase-bright sphere cells found floating around the fragments, attached on culture flasks. After 20 days, a specific type of cells, predominantly ellipsoids or fusiform cells was characterized in vitro. The indirect immunofluorescence examination showed cells expressing markers of neuronal precursors (GFAP, neurofilament, oligodendrocyte, and III â-tubulin). The cell proliferation index showed Ki67 immunostaining with a trend to label cell groups throughout the apical region, while PCNA immunostaining label predominantly cell groups lying above the basal lamina. The transmission electron microscopy from the olfactory epithelium of dogs revealed cells with electron-dense cytoplasm and preserving the same distribution as those of positive cell staining for PCNA. Metabolic activity was confirmed by presence of euchromatin in the greatest part of cells. All these aspects give subsidies to support the hypothesis about resident progenitor cells among the basal cells of the olfactory epithelium, committed to renewal of these cell populations, especially neurons.

A histological and histochemical study of the oesophagus and oesogaster of the Senegal sole, Solea senegalensis.

A histological and histochemical study was performed in the buccal cavity and papillae, which were around the teeth, as well as in the oesophagus and oesogaster of the Senegal sole, Solea senegalensis adult specimens. The oesophagus and oesogaster were made up of four distinct layers: mucosa, submucosa, muscular and serous. Two morphological types of epithelial cells were distinguishable in the oesophageal mucosa: the more numerous type cells possessed an electron-dense cytoplasm, whereas the cytoplasm was electron-clear in the other cells. Mucus-secreting cells were the dominant feature of the epithelium throughout the oesophagus. These goblet cells were filled with numerous mucous droplets of low electron-density. The oesophagus was devoid of taste buds. In the oesogaster mucosa, three types of cells were distinguished: dark, rodlet and light epithelial cells. Dark epithelial cells showed different characteristics from that in the oesophagus: the nucleus was irregular with an electron-dense hyaloplasm, the cytoplasm had a scarce smooth and granular endoplasmic reticulum; a Golgi apparatus consisted of four parallel cisternae, dense granules without membrane, lysosomes and numerous mitochondria. The rodlet cells were elongated, contained rod-like structures and were surrounded by an electron-dense capsule-like structure. The bulk of the rodlet cell was composed of up to 20 extended rodlet units. Light epithelial cells of the oesogaster had the same characteristics as those observed in the oesophagus and contained numerous mitochondria with a dense matrix, abundant smooth endoplasmic reticulum and numerous vesicles. In the goblet cells of the papillae, sulfomucin was recognised, since they showed alcianophilia (alcian blue pH 1.0 and 0.5). These cells were negative to protein reaction (bromophenol blue) and contained -S-S- and SH groups. Enzymatic activities (alkaline phosphatase, acid phosphatase, ATPase (pH 7.2 and 9.4) and lipid reactions were negative in the goblet cells of the buccal cavity. Epithelial cells of oesophagus contained a weak presence of acid and neutral mucopolysaccharides. Oesophageal goblet cells contained carboxylated, sulphated (weakly and strongly ionised) mucosubstances and sialic acid. Most goblet cells did not contain proteins and presented disulphide (-S-S-) and sulphydril (-SH) groups. Proteins in general, and in particular those rich in lysine, tyrosine and arginine were present in the epithelium, lamina propria, submucosa and muscular layer of the oesophagus. Lipids in general and phospholipids were observed in the oesophageal epithelium while unsaturated, acid and neutral lipids were not observed. The lamina propria and submucosa contained a weak presence of phospholipids and unsaturated lipids. Acid phosphatase and ATPase (pH 7.2) activities were observed in the lamina propria, submucosa and muscular regions, while ATPase (pH 9.2) activity was weak in these areas. ATPase activity (pH 7.2 and 9.5) was very weak in the epithelium. Oesophageal goblet cells were negative to lipid and enzymatic reactions.

Ultrastructure and cytochemical localization of acid phosphatase of laticifers in Euphorbia kansui Liou

Acid phosphatase (AcPase) activities are involved in the degeneration process of cytoplasm in plants. In this study, acid phosphatase was detected by the method of lead nitrate and cytochemical electron microscopy during the development of nonarticulated laticifers in Euphorbia kansui Liou. The most important feature in the differentiation of the laticifers in E. kansui is that the development of small vacuoles arises from endoplasmic reticulum (ER). The mature laticifers possess a thin layer of electron-dense peripheral cytoplasm in which the organelle cannot be distinguished and a large central vacuole filled with latex particles. AcPase cytochemistry studies show AcPase reaction products congregated into heaps are distributed along the tonoplast of central vacuole and around the mitochondria and plastids. Some small vacuoles which develop at later developmental stages of laticifers contain AcPase reaction products. As a result, the central vacuole is formed by cellular autophagy and fusion of small vacuoles which apparently arises from ER.

Distribution and three‐dimensional appearance of the interstitial cells of Cajal in the rat stomach and duodenum

The relationship between the interstitial cells of Cajal (ICC) and enteric nerves or smooth muscles cells is not fully defined. Presently, distribution and appearance of ICC in the rat stomach and duodenum was studied by immunohistochemistry, electron microscopy, and three-dimensional reconstruction. c-kit expressing ICC were regularly observed in the Auerbach's myenteric plexus (AP) of the stomach and duodenum. ICC in stomach and duodenum muscle layers was dissimilarly distributed. c-kit immunoreactive cells were sparsely distributed in the stomach circular muscle layer but were abundant in the duodenum deep muscular plexus (DMP). Electron microscopy revealed that stomach ICC-AP were irregular ovals with few cytoplasmic processes, and possessed an electron-dense cytoplasm, numerous mitochondria, intermediate filaments, and caveolae. Duodenum and stomach ICC-AP were similar in appearance. Ultrastructure observations and three-dimensional reconstructions revealed ICC-AP processes wrapping the nerve fibers and projecting into the space between smooth muscle cells. While ICC-AP was occasionally close to enteric nerves or smooth muscle cells, no connections were observed. ICC-DMP in duodenum was elongated and adopted the same cell axis orientation as the circular muscle cells. Unlike ICC-AP, ICC-DMP formed gap junctions with smooth muscle cells and had close contact with nerves. These results indicate that ICC-AP is regularly distributed in stomach and duodenum, while ICC-DMP is exclusively located in the duodenum. ICC-DMP, which possess gap junctions and closely contacts nerves, may participate in neuromuscular transmission.

Ultrastructural changes of the olfactory bulb in manganesetreated mice

The effect of manganese toxicity on the ultrastructure of the olfactory bulb was evaluated. Male albino mice were injected intraperitoneally with MnCl2 (5 mg/Kg/day) five days per week during nine weeks. The control group received NaCl (0.9%). The olfactory bulbs of five mice from each group were processed for transmission electron microscopy after 2, 4, 6 and 9 weeks of manganese treatment. On week 2, some disorganization of the myelin sheaths was observed. After 4 weeks, degenerated neurons with dilated cisternae of rough endoplasmic reticulum and swollen mitochondria appeared. A certain degree of gliosis with a predominance of astrocytes with swollen mitochondria, disorganization of the endomembrane system, dilation of the perinuclear cisternae and irregularly shaped nuclei with abnormal chromatin distribution were observed after 6 weeks. Some glial cells showed disorganization of the Golgi apparatus. On week 9, an increase in the number of astrocytes, whose mitochondrial cristae were partially or totally erased, and a dilation of the rough endoplasmic reticulum were found. Neurons appear degenerated, with swollen mitochondria and a vacuolated, electron dense cytoplasm. These changes seem to indicate that the olfactory bulb is sensitive to the toxic effects of manganese.

Cell wall metabolism during ripening and storage of nonclimacteric sour cherry (Prunus cerasus l., cv. Kántorjánosi)

Ripeness: The colour of green (ripeness stage I), pit hardening (II), light red (a, b) (III, IV), red (V) and dark red (VI) sour cherry fruits ( Prunus cerasus cv. Kantorjanosi) were characterised by CIELAB L*, a* and b* values. L* and b* decreased as a function of ripeness, a* intensively increased between green (I) and pit hardening (II) stages. The cell wall of green fruit was intact, but the electron dense cytoplasm concentrated along the cell wall and showed a number of degradation signs. The pit hardening stage (II) resulted in more structural break down in the cytoplasm and in the cell wall. Large numbers of plastoglobuli were in the plastids resulting in chloroplast-gerontoplast conformation. The most striking feature of light red fruits is the dissolution of the walls. Middle lamellae almost completely disappeared. In ripe fruits, the wall degradation was even more prominent. The regular structure of the cytoplasm had almost completely disappeared. The total pectin content between pit hardening a...

OSOBENNOSTI STRUKTURNOY ORGANIZATsIISUSTAVNOGO KhRYaShchA v zavisimosti ot stadii gonartroza

The purpose - revealing of features of the structural organization of the articulate cartilage of a knee joint at patients with various stages of arthrosis. Materials and methods. Morphological researches of an articulate cartilage are visited at 52 patients with various stages of gonarthrosis. The control group was made by 5 men and 5 women in the age of from 18 till 25 years with traumatic damages of components of a knee joint. The basic group was made by 42 persons. On a stage of gonarthrosis patients have been divided into 2 subgroups: 23 patients with 1-2 stage and 19 patients with 2-3 stage of gonarthrosis. The histological material for research has been received at performance medical-diagnostic arthroscopies a knee joint. Results. It is noted, that morphological changes in an articulate cartilage at gonarthrosis are connected with change of density intercellular matrix and, depending on a stage of process, of chondrocyte dystrophy, necrosis and apoptosis are took place. In cytoplasm of chondrocyte there is a decrease in concentration of cytoplasmic organelles, lipids accumulation and electron density inclusions. Conclusion. The most expressed structural changes in the articulate cartilage, connected with local destruction of chondrocytes, lipids and electron density inclusions accumulation are noted at patients with 2-3 stage of gonarthrosis.

Ultrastructure of the digestive system and the fate of midgut during embryonic development in Porcellio scaber (Crustacea: Isopoda)

Microscopic anatomy of the digestive system in embryos and larvae of the terrestrial isopod crustacean Porcellio scaber was investigated by light bright field, fluorescence and electron microscopy. During marsupial ontogenetic development the event-dependent staging was used to discriminate the various embryonic stages. At the late embryo stage the differentiation of the ectodermal part of the gut into the complex filtering foregut and the hindgut with absorptive and transporting functions is accomplished. The gut of the marsupial manca larva is fully developed and similar to that of the adult. In early embryos the endodermal midgut gland primordia are filled with yolk and lipid globules. In late embryos the epithelium of paired midgut gland tubes is composed of two cell types; one of them exhibits orange autofluorescence. The endodermal cells located between the foregut and the midgut glands of late embryos form the prospective midgut. The cells have electron dense cytoplasm, abundant glycogen fields, endoplasmic reticulum, dictyosomes and numerous vesicles. In the adults the endodermal cells of the midgut remain only in the midgut gland ducts which connect the midgut glands and the foregut. Details of the cellular ultrastructure and morphogenesis of the ectodermal and endodermal parts of the digestive system during embryonic development of Porcellio scaber provide data for further phylogenetic and comparative studies in peracaridan crustaceans and other arthropods.

Effect of Hypodynamy on Structure and Alkaline Phosphatase Activity of Kidney in Japanese Quails

The objective of the study was to observe the effect of experimental hypodynamy simulating weightlessness in space on the structure, ultrastructure and alkaline phosphatase activity of kidney in Japanese quail (Coturnix coturnix japonica). Two days after hatching, the quails were suspended in special shirts below the cage ceiling so their feet did not touch the floor. They could consume food and water ad libitum. Experimental animals were sacrificed after 14, 21, 28, 35, 42, 49 and 56 days of hypodynamy. Birds of the same age, hatched at the same time, and fed the same diet were used as a control. Samples of kidney were processed for light (LM) and transmission electron microscopy (TEM), and alkaline phosphatase (AP) analysis. Short-term (14–28 days) hypodynamy caused no marked damage to the structure and ultrastructure of kidneys. However, after long-term (35–59 days) hypodynamy, morphological changes were observed in some cells of the proximal and distal tubules. The dying cells in proximal tubules, observed in semi-thin sections by LM, were dark and contained a nucleus of irregular shape. Observation by TEM showed that their nucleus was dark and shrivelled and the electron-dense cytoplasm contained long, dense, rod-shaped mitochondria with thin mitochondrial cristae. Microvilli were present on the apical surface of cells and formed a brush border. Sporadic dying cells were also observed in distal tubules. Large, light vacuoles were found in the cytoplasm of cells of collecting tubules, however, the structure of renal corpuscles and medullary loops remained undisturbed. Microscopical analysis by means of a direct TUNEL reaction on days 35 to 59 of hypodynamy showed a moderate occurrence of cellular apoptosis in the proximal and distal tubules of experimental Japanese quail. The activity of AP in the brush border of the proximal tubules on days 14–29 of hypodynamy was normal in experimental animals and showed no significant differences in comparison with the control. Between days 35 and 59 of hypodynamy a moderate decrease in the content of AP in experimental animals in comparison with the control (P < 0.01) was observed. Our results indicated that long-term hypodynamy caused apoptosis of some cells in the proximal and distal tubules and therefore had a negative effect on the kidney structure. The occurrence of apoptosis was only sporadic. We presume no negative effect on the kidney function and the Japanese quails are capable of further development under the conditions of prolonged simulated weightlessness.

Formation of striatum structural and ultrastructural organization in rat postnatal ontogenesis at changes of conditions of their embryonic development

Using light microscopy (Nissl and Golgi techniques), electron microscopy and immunohistochemistry, formation of structure of the brain striatum dorsolateral part from birth to three month of age was studied in rats submitted to acute hypoxia at the period of embryogenesis. Hypoxia at the 13.5th day of pregnancy (E 13.5) was found to lead to a delay of neuronogenesis for the first two weeks of postnatal development as compared with control animals, and the majority of large neurons for this period were degenerated by the type of chromatolysis with swelling of the cell body and processes and lysis of cytoplasmic organelles. By the end of the third week, shrunken hyperchromic or pycnomorphic neurons with the electron-dense cytoplasm and enlarged tubules of endoplasmic reticulum and Golgi complex were also observed. An increased number of swollen processes of glial cells was found in neuropil around the degenerating neurons. By the 30th day as well as in adult animals, destruction of mitochondrial apparatus, an increased number of lysosomes, and blade-shaped nuclei, which are characteristics of the apoptotic cell death, were observed. This is also confirmed by an increased expression of proapoptotic protein (p53) and its co-localization with caspase-3 in a part of neurons. Morphometric analysis showed a decrease of the cell distribution density in striatum and a change of ratio of different cell types in hypoxia-exposed rats as compared with control group. The most pronounced decrease (42.3% at the 5th day, 14.2% at the 10th day, p < 0.01) of the number of large neurons (larger than 80 μm2) was revealed at early stages of postnatal ontogenesis. After 3 postnatal weeks, the number of middle-sized neurons (30–95 μm2) decreased (by 11.8–19.2% as compared with control, p < 0.05). The obtained data have shown that changes of embryogenesis conditions (hypoxia) at the period of the most intensive proliferation of telencephalon neuroblasts lead to impairment of the process of striatal nervous tissue formation. This might be the cause of delay of development and disturbances of behavior and learning, which are observed in rats exposed to prenatal hypoxia.

Ultraestrutra do epitélio pigmentar da retina e dos cones do peixe Antártico Notothenia coriiceps Richardson submetido à luz e ao escuro

The Antarctic fish Notothenia coriiceps Richardson, 1844 lives in an environment of daily and annual photic variation and retina cells have to adjust morphologically to environmental luminosity. After seven day dark or seven day light acclimation of two groups of fish, retinas were extracted and processed for light and transmission electron microscopy. In seven day dark adapted, retina pigment epithelium melanin granules were aggregated at the basal region of cells, and macrophages were seen adjacent to the apical microvilli, between the photoreceptors. In seven day light adapted epithelium, melanin granules were inside the apical microvilli of epithelial cells and macrophages were absent. The supranuclear region of cones adapted to seven day light had less electron dense cytoplasm, and an endoplasmic reticulum with broad tubules. The mitochondria in the internal segment of cones adapted to seven day light were larger, and less electron dense. The differences in the morphology of cones and pigment epithelial cells indicate that N. coriiceps has retinal structural adjustments presumably optimizing vision in different light conditions.

Fine structure of the retinal pigment epithelium and cones of Antarctic fish Notohenia coriiceps Richardson in light and dark-conditions

The Antarctic fish Notothenia coriiceps Richardson, 1844 lives in an environment of daily and annual photic variation and retina cells have to adjust morphologically to environmental luminosity. After seven day dark or seven day light acclimation of two groups of fish, retinas were extracted and processed for light and transmission electron microscopy. In seven day dark adapted, retina pigment epithelium melanin granules were aggregated at the basal region of cells, and macrophages were seen adjacent to the apical microvilli, between the photoreceptors. In seven day light adapted epithelium, melanin granules were inside the apical microvilli of epithelial cells and macrophages were absent. The supranuclear region of cones adapted to seven day light had less electron dense cytoplasm, and an endoplasmic reticulum with broad tubules. The mitochondria in the internal segment of cones adapted to seven day light were larger, and less electron dense. The differences in the morphology of cones and pigment epithelial cells indicate that N. coriiceps has retinal structural adjustments presumably optimizing vision in different light conditions.

Morphological Evidence that Xenon Neuroprotects against N‐Methyl‐dl‐Aspartic Acid‐Induced Damage in the Rat Arcuate Nucleus

The hyperactivation of glutamate receptors, especially those of the N-methyl-d-aspartate subtype (NMDA), can induce excess calcium entry into cells, leading to neuronal death. Since the anesthetic gas xenon behaves as an NMDA antagonist, the present article investigated, by distinct morphological approaches and after different times, the possible neuroprotectant effects of this gas in a model of neuronal damage induced by N-methyl-dl-aspartic acid (NMA) on rat arcuate nucleus. Rats were assigned to the following groups: controls; xenon exposure; NMA treatment; or xenon exposure + NMA treatment. Animals were placed in an experimental cage and after 10 min a mixture of xenon (or nitrogen) 70% and oxygen 30% was delivered. After 3 h, 1, 2, 5, or 7 days from gas exposure, rats were euthanized and the whole brain was removed and processed for either transmission electron microscopy or light microscopy. In the arcuate nucleus from NMA-treated animals only 40-60% of cell population survived in all times with several degenerating neurons giving the typical appearance of a "bull's eye." At ultrastructural level, chromatin margination, nuclear shrinkage, mitochondria with matrix dilution, dilated endoplasmic cisternae, and electrondense cytoplasm were detected. Xenon alone did not induce changes, but reduced of about 50% NMA-induced cell loss as well as degenerating neurons, with the maximal neuroprotection at 7 days. These results confirm that in the rat arcuate nucleus NMA can induce a severe neuronal damage that is already marked after 3 h. Xenon significantly reduced the neuronal damage at all times and can be then regarded as a promising neuroprotectant agent.

Injury of learning and memory induced by brain aging and the relationship with the changes of myelin structure

目的 探讨脑老化时认知功能改变、髓鞘结构和成分变化及其之间关系.方法使用穿梭箱、电镜和免疫组织化学技术,观察了老年大鼠学习、记忆维持功能、髓鞘和轴突的超微结构改变,以及髓鞘碱性蛋白表达变化.结果在训练第1天至第6天,老年大鼠主动逃避反应率(AAR)、总逃避反应(GAR)率较青年组显著减慢(P <0.01),逃避潜伏时(LER)较青年组延长(P <0.01),训练第6天青年组GAR达到(89.0±6.6)%,老年只有(40.5±6.8)%,青年组LER为(3.16±0.47)s,老年组为(6.10±0.71)s.在记忆功能维持上,训练后第5天和第10天,老年组大鼠AAR、GAR很快降低,LER明显升高,较青年组差异有显著性(P <0.01).老年大鼠髓鞘异常为轴突周围间隙扩大,髓鞘致密层分离,部分板层结构破坏,髓鞘内形成大泡,轴突直径增加,轴突内含有吞噬小泡、致密颗粒和巨大线粒体,少突胶质细胞电子密度增加.老年大鼠髓鞘碱性蛋白(MBP)表达下降.结论老年大鼠学习及记忆维持能力降低,老化可以导致轴突及髓鞘的结构变化,MBP表达降低,髓鞘结构和成分改变可能参与老化时学习和记忆损害。

Structure of Bovine Fungiform Taste Buds and Their Immunoreactivity for Gustducin

The taste buds of bovine fungiform papillae were studied by light and electron microscopy using both histological and immunohistochemical methods. The taste buds existed in the epithelium of the apical region of the papillae. By electron microscopy, two types of taste cells, namely type I and type II cells, could be classified according to the presence of dense-cored vesicles, the cytoplasmic density and the cell shape. Type I cells were thin, had an electron-dense cytoplasm containing dense-cored vesicles, and possessed long thick apical processes in the taste pore. Type II cells were thick, had an electron-lucent cytoplasm containing many electron-lucent vesicles, rather than dense-cored vesicles, and possessed microvilli in the taste pore. Immunohistochemical staining with an antiserum against gustducin was investigated by both light and electron microscopy using the avidin-biotin complex (ABC) method. Some, but not all, of the type II cells exhibited gustducin immunoreactivity, whereas none of the type I cells showed any immunoreactivity.

Evidence of apoptotic effects of 2,4-D and butachlor on walking catfish, Clarias batrachus, by transmission electron microscopy and DNA degradation studies

Apoptosis or programmed cell death is characterized morphologically by chromatin condensation, cell shrinkage, fragmentation of the nucleus and cytoplasm, and consequently formation of apoptotic bodies. It has also been best characterized by the cleavage of DNA into nucleosomal size fragments of 180–200 bp or multiples of the same. Contrary to this, under extreme conditions, the cells were found to show adaptive response to apoptosis and unable to regulate their own death; necrosis is therefore predominantly observed. In the present study, we showed induction of apoptosis in Clarias batrachus due to sublethal concentration of 2,4-D and butachlor at multiple exposure time. The first phase of the study involved light microscopy (LM) and transmission electron microscopy (TEM) for ultrastructural abnormalities of the germinal tissues. While, in the second phase of the study, DNA degradation of blood and hepatic tissue was resolved on agarose gel electrophoresis. In histopathological studies, large numbers of stage II oocytes were noted for nuclear blebbing irrespective of the test chemical. Some of the butachlor-exposed oocytes showed vacuolation and electron dense cytoplasm along with thickened nuclear envelope, having close association with the lysosomes on the cytoplasmic side. Some oocytes undergo nuclear blebbing having inner dense core and translucent cytoplasm. Leydig cells were slightly hypertrophied and few appeared pycnotic, a process involving necrotic changes in which the cell nuclei were characterized by rounding up and condensation resulting in hyperchromatic staining or pycnosis. In testicular tissue, spermatogonial nuclei had irregular large clumps of heterochromatin adjoining the nuclear membrane indicating initial stage of apoptotic cell death. Electrophoretic separation resulted in a ladder pattern of blood DNA and smear like pattern of hepatic DNA. These results indicate that the above herbicides are able to induce apoptosis both at molecular as well as cytological level. A reference dose or safety factor approach to calculate risk of human exposure to both chemicals is still awaited.

Ultrastructural evaluation of the radioprotective effects of melatonin against X‐ray‐induced skin damage in Albino rats

Our knowledge about the radioprotective effects of melatonin against X-ray-induced skin damage is still lacking. To examine these effects, an animal model of 60 Albino rats was used. The animals were divided into five groups: Group 1, nonirradiated; Group 2, X-ray irradiated (XRI, 8 Gy); Group 3, XRI pretreated with solvent (ethanol and phosphate-buffered saline); Group 4, nonirradiated group treated with melatonin; and Group 5, XRI pretreated with melatonin. The skin was evaluated for ultrastructural changes using transmission electron microscopy (TEM). When compared to the nonirradiated skin (Groups 1 and 4), XRI skin (Groups 2 and 3) showed features of both cell injury and increased metabolic activity. The former included changes such as condensation of the nuclei, vacuolization of the cytoplasm, dilatation of the rough endoplasmic reticulum, swelling of the mitochondria with cristolysis, destruction of the ribosomes and intermediate filaments, fragmentation of the keratohyaline granules and loss of the irregularity of the basal cell borders. The central cells of the sebaceous gland alveoli had larger irregular nuclei and few lipid droplets in their cytoplasm. The hair follicle cells had heterochromatic nuclei and less electron dense cytoplasm containing few complements of the organelles. The features of increased metabolic activity included increased euchromatin, irregularity of the nuclear membrane and increased branching of the melanocytes. Also, an increased number of the Birbeck granules were seen in the Langerhans cells. When compared to the irradiated skin (Groups 2 and 3), these changes were mild or absent in the skin of XRI animals pretreated with melatonin (Group 5). The ability of melatonin to minimize the injurious effects of XRI suggests a radioprotective role. The clinical ramifications of these observations warrant further studies.

Long-term effect of pulsed Nd:YAG laser irradiation on cultured human periodontal fibroblasts

The purpose of this study was to investigate the long-term effect of Nd:YAG laser irradiation on cultured human periodontal fibroblasts (hPF). The cultured hPF were irradiated by pulsed Nd:YAG laser. The power delivery was 50 mJ x 10 pps (pulse per second) with irradiation duration 60, 120, 180, or 240 seconds. The viability and collagen content of laser-irradiated hPF were assessed on day 5 after laser treatment. Light microscope and transmission electron microscope (TEM) were used to observe cytomorphological change. The irradiated hPF cultured in mineralizing medium for 28 days were examined by alizarin red S and Von Kossa stain. The cellular viability and collagen content of hPF decreased after Nd:YAG laser irradiation. Cell damage was noted with retraction of cellular processes, loss of normal architecture, and lysis of some cells. However, survived hPF proliferated and migrated to the cell-debris-associated deposits. The electron-dense cytoplasm and amorphous organelles in laser-damaged cells was revealed by TEM. In vitro mineralization was demonstrated in the long-term laser-irradiated hPF cultured in mineralizing medium. Nd:YAG laser irradiation induced partial loss of cellular viability and collagen content. The co-existence of viable cells and progressive degeneration of laser-damaged cells was associated with the in vitro mineralization of hPF.

Lectin Histochemistry of Taste Buds in the Circumvallate Papilla of the Rat

In mammals, taste buds are localized mostly within two regions, i.e. lingual and palatal epithelium. In the lingual epithelium, taste buds are localized in three lingual papillae; fungiform, foliate and circumvallate papillae. In the palatal epithelium, taste buds are present in the nasoincisor papillae and soft palate. In addition, few taste buds are recognized at the laryngeal surface of epiglottis. Taste buds contain 50–80 elongated specialized epithelial cells (taste cells) and proliferative basal cells (progenitor cells). Ultrastructually, taste cells have traditionally been classified into two types of cells; dark and light cells. Dark cells or type I cells have an electron-dense cytoplasm and thought to be supporting cells. Light cells are characterized by the presence of electron-lucent cytoplasm and further subdivided into two types based on the presence of synaptic vesicles. Light cells without apparent synaptic vesicles are called type II cells, while those with synaptic vesicles are termed as type III cells. Progenitor cells are located at the basal portion of the taste buds. There are at least two theories for cell lineage of the taste buds; each type of cells in taste buds has individual progenitor cells (multi cell line theory) and all types of cells are derived from the same progenitor cell (one cell line theory). Several histochemical studies to demonstrate the expression of bioactive substances in specific cell type of taste cells for the analysis of cell lineage of the taste buds. Lectins are useful for histochemistry and have been used in several areas to identify particular cell populations on the base of different ability to bind with high specificity to different carbohydrate epitopes of cell membrane. Although several lectin histochemical studies have been carried out to the taste buds to map the overall distribution of different kinds of lectins in the different location of taste buds in various species (Witt and Reutter, 1988; Witt and Miller, 1992; Zeng et al., 1995; Ohnishi et al., 2000; Kano et al., 2001), few studies are conducted on the relation between lectin binding patterns and specific cell types within the taste buds. The present presentation focused on the lectin binding pattern of rat circumvallate papilla with special references to the relation with type II cells, i.e. α-gustducin-immunoreactive cells. Ulex europaeus agglutinin-I (UEA-I) reacts strongly with α(1, 2)linked fucose. In the taste buds of the circumvallate papilla, UEA-I was found to bind with the cell membrane of taste buds (Figure 1A, compared with Figure 1C). Within the taste buds, UEA-I bound to almost all individual cells in the taste buds. Most blood group H type 2 antigen (AbH) immunoreactive cells co-expressed UEA-I (Figure 1B). In addition to the taste buds, UEA-I binds occasionally to the keratinized layer of the trench wall of the circumvallate papilla and in the lingual epithelium. Although lectin from Lotus tetragonolobus is also specific for L-fucose, it did not, however, bind to the taste bud cells. Peanut agglutinin (PNA) is specific for terminal β-galactose and binds to the membrane of rounded-shaped cells at the basal portion of taste buds. Spindle-shaped cells within the taste buds were devoid of PNA binding. In the trench wall epithelium, PNA strongly labeled the membrane of cells from basal cell layer to granular cell layer. Cells at keratinized cell layer lack PNA bindings. In the lingual epithelium, strong bindings for PNA were also detected from basal cell layer to granular cell layer. Jacalin is purified from jackfruit and specific for D-galactose. Similar to PNA, jacalin binds the membrane of cells located from basal cell layer to granular layer in the trench wall epithelium. Keratinized cells lacked in jacalin binding. Some rounded cells at the basal portion of the taste buds were also found to bind to jacalin. PNA bound to membrane of some rounded-shaped cells that does not react with jacalin. Within the taste buds, some spindle-shaped cells reacted with both wheat germ agglutinin (WGA) and succinyl WGA. The number of cells binding to WGA is slightly smaller than that binding to succinyl WGA. Many cells binding to WGA and succinyl WGA displayed immunoreactivity for α-gustducin; Not all cells lebeled with WGA or succinyl WGA showed α-gustducin immunoreactivity and vise versa. Dolichos bifforus agglutinin (DBA) binds to the mostly apical portion of the spindle-shaped cells within the taste buds of circumvallate papilla. Occasionally, DBA also binds to the cytoplasm of the basal portion of taste buds. The number of cells binding to DBA is low compared to cells binding to WGA or succinyl WGA. Most of cells binding with DBA did not show immunoreactivity for α-gustducin. The present lectin histochemical analysis of taste cells showed that some lectins bind specifically to certain types of cells in the rat circumvallate papilla.