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.

The 12th China-Japan Joint Seminar on Histochemistry and Cytochemistry

01 Differentiation-dependent changes in DNA methylation and histone H3 acetylation: possible roles of these epigenetic factors in mouse spermatogenesis Takehiko Koji Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan Mammalian spermatogenesis is a well-ordered process, consisting of spermatogonial mitosis, spermatocytic meiosis and spermiogenesis.The process is also characterized by successive chromatin condensation and a high frequency of spermatogenic cell apoptosis.Therefore, epigenetic factors can be implicated in the regulation of spermatogenesis and any epigenetic disorder may lead to the induction of apoptosis. When we analyzed global level of DNA methylation by immunohistochemistry with anti-5-methylcytosine in paraffin-embedded sections of mouse testis, there was no difference among any stages of germ cells.

ULTRASTRUCTURAL CYTOCHEMISTRY OF HUMAN GASTRIC CANCER: ELECTRON MICROSCOPIC OBSERVATIONS OF FIVE ORGANELLAE MARKER ENZYMES

The distribution of ALPase, ACPase, G6Pase TPPase and CCOase of gastric cancer and normal gastric epithelium were studied ultrastructurally. The results showed that normal gastric epithelium had no ALPase reaction. The reactions of ACPase, G6Pase, TPPase and CCOase were found in the corresponding organellae which were cosistent with their functions. In tubular adenocarcinoma cells, their reactions were more apparent in the corresponding organellae. Some cells of tubular adenocarcinomas showed ALPase reaction. The mucinous adenocarcinoma cells had higher ACPase and TPPase reactions. In poorly differentiated adenocarcinoma cells, the five marker enzymes showed negative or faint reactions. The biological significance and mechanisms of distribution of the five marker enzymes were discussed.

Cytochemical localization of ATPase and sub-cellular variation in mesophyll cell of Cyclocarya paliurus seedlings under iso-osmotic stress and calcium regulation

The ultrastructural distribution and active location of ATPase and the ultrastructural variations were investigated in mesophyll cells of Cyclocarya paliurus seedlings after iso-osmotic salt/water treatments in combination with calcium regulation. C. paliurus seed- lings were treated with five groups （control, 85 mM NaCl, 85 mM NaCl＋ 12 mM Ca（NO3）2, PEG iso-osmotic to 85 mM NaCl and PEG iso-osmotic to 85 mM NaCl＋12 mM Ca（NO））2） in a hydroponic system in a phytotron. Results show that under normal growth conditions the ATPase activity was low and the enzyme was primarily located on the nucleus. After 12 days of iso-osmotic salt/water treatments, ATPase activity on the tonoptast increased. Osmiophilic globules for iso-osmotic water treatment were greater than that for iso-osmotie salt treatments. The ATPase activity increased and was mostly transferred onto the nucleus for calcium regulation treatment under iso- osmotic salt/water stresses, and the osmiophilic globules significantly decreased under iso-osmotic water stress with calcium regulation. The ATPase located on the nucleus indicated that the degree of salt/drought damage that seedlings suffered was slighter, while the amount of the enzyme located on the tonoplast showed that the degree of salt/drought damage there was more serious. After 4 and 20 days of iso- osmotic treatments, the injury suffered by the leaf ultrastructures of C. paliurus seedlings for iso-osmotic treatment with calcium regula- tion was lower than those without calcium regulation, especially for the iso-osmotic water treatments. Preliminary analysis suggests that the iniury suffered by C. paliurus seedlings was lower for iso-osmotic salt treatments than for iso-osmotic water treatments, while the effect of calcium regulation under iso-osmotic water stress was greater than that of the iso-osmotic salt stress.

Ca^(2+) cytochemical changes of hepatotoxicity caused by halothane and sevoflurane in enzyme-induced hypoxic rats

AIM： To investigat the relation between hepatotoxicity of halothane and sevoflurane and altered hepatic calcium homeostasis in enzyme-induced hypoxic rats. METHODS： Forty-eight rats were pretreated with phenobarbital and randomly divided into six groups （eight in each group） and exposed to O2/N2/1.2 MAC anesthetics for 1 h： normal control （NC）, 21% O2/79% N2; hypoxic control （HC）, 14% O2/86% N2; normal sevoflurane （NS）, 21% O2/ N2/1.2MAC sevoflurane; hypoxic sevoflurane （HS）, 14% O2/N2/1.2MAC sevoflurane; normal halothane （NH）21%O2/79%N2/1.2MAC halothane; hypoxic halothane （HH）, 14%O2/N2/1.2MAC halothane. Liver specimens and blood were taken 24 h after exposure to calcium and determined by EDX microanalysis. RESULTS： The liver of all rats given halothane （14% O2） had extensive centrilobular necrosis and denaturation. Morphologic damage was accompanied with an increase in serum glutarnic pyruvic transminase. In groups NH and HH, more calcium was precipitated in cytoplasm and mitochondria. CONCLUSION： These results suggest that halothane increases cytosolic Ca^2＋ concentration in hepatocytes. Elevation in Ca^2＋ concentration is implicated in the mechanism of halothane-induced hepatotoxicity. sevoflurane is less effective in affecting hepatic calcium homeostasis than halothane.

Cytomorphology and Cytochemical Localization of K-type and T-type Cytoplasmic Male Sterile Pollens in Wheat

By using the technology of microscopy, electron microscopy and enzyme cytochemical localization , comparison was made of the cytomorphology, ATPase and cytochrome oxidase activity in pollens during microspore formation and pollen development, for K-type cytoplasmic male sterile(CMS)lines,T-type CMS lines and their maintainer lines on wheat. The results indicated that pollen abortion of the T-type CMS lines mainly occurred at the later mononucleate pollen stage, and cytomorphological changes of the abortive pollen began with a vacuolar membrane, and pollen aborting was related to a lack of ATPase activity in the nucleus and nucleolus during the mononucleate pollen stage. Pollen abortion of the K-type CMS lines took place mainly during the later binucleate and trinucleate stages, and cytomorphological changes of the abortion first began with mitochondria. The abortion was related to intine disruption.

Exfoliative Cytology of Oral Mucosa Epithelium: Cytochemical Study and Morphologic Analysis of Patients with Type 2 Diabetes

Background: Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia. An association between diabetes mellitus and periodontal disease has been reported. However, few comprehensive reports have examined the impact of chronic hyperglycemia on oral mucosal epithelial cells. This study clarified the effect of diabetes on oral mucosal epithelium cells. Methods: Cells from 44 patients with type 2 diabetes and 10 healthy controls were collected from the buccal mucosa, gingiva, and lateral margin of the tongue by exfoliation. Samples were subjected to Papanicolaou staining;immunochemical staining using anti-AGE antibody, anti-Ki-67 antibody, and anti-p53 antibody;and lectin chemical staining. Furthermore, the nuclear area and cytoplasmic area were measured, and the nuclear/cytoplasm ratio was calculated. Results: A chromatin condensation-like substance was found in samples from some diabetic patients. In AGE staining, positive cells were found in both groups, although the rate of positive cells tended to be higher in diabetic patients. No positive findings were found for Ki-67 and p53 antibodies. PNA and RCA120 showed differences in staining between diabetic patients and controls. The nuclear area was significantly expanded in diabetic patients (P < 0.05). There was no significant difference between groups in the cytoplasmic area. The nuclear/cytoplasm ratio was significantly increased in diabetic patients (P Conclusion: These findings suggest that in patients with persistent hyperglycemia, changes in the sugar chain terminal of the cell surface may influence mucosal epithelial cell differentiation. The appearance of chromatin-like substances may be an indicator of the need for better management of type 2 diabetes.

Cytochemical characterization of yolk granule acid phosphatase during early development of the oyster Crassostrea gigas(Thunberg)

In this study, a cytochemical method and transmission electron microscopy was used to examine acid phosphatase activities of yolk granules throughout the early developmental stages of the Pacific oyster Crassostrea gigas. This study aimed to investigate the dynamic change of yolk granule acid phosphatase, and the mechanisms underlying its involvement in yolk degradation during the early developmental stages of molluscs. Three types of yolk granules （YGI, YGⅡ, and YGⅢ） that differed in electron density and acid phosphatase reaction were identified in early cleavage, morula, blastula, gastrula, trochophore, and veliger stages. The morphological heterogeneities of the yolk granules were related to acid phosphatase activity and degrees of yolk degradation, indicating the association of acid phosphatase with yolk degradation in embryos and larvae of molluscs. Fusion of yolk granules was observed during embryogenesis and larval development of C. gigas. The fusion of YGI （free of acid phosphatase reaction） with YGII （rich in acid phosphatase reaction） could be the way by which yolk degradation is triggered.

The effects of inosine and Ligusticum wallichii on ischemic injury of renal proximal tubule epithelial cells:cytochemical study

Cytochemical methods were used to evaluate the changes of intracellularcalcium,plasma membrane permeability and their relationship during ischemia andreflow in rat renal proximal tubule epithelial cells.In addition,the preventive ef-fects of inosine and Ligusticum wallichii on ischemia and reperfusion cell injurywere studied.The results showed that the preventive effects of inosine andLigusticum wallichii were obvious.Redistribution and slight increase of calciumoccurred during the ischemic period in the proximal tubule cells.In this periodthe changes of plasma membrane permeability were gentle.Following the exten-sion of reflow time,a large amount of calcium deposited in the cells andmitochondria.The plasma membrane became broken.There was a positivecorrelation between the calcium level in mitochondria and that in cytoplasm.

AN ENZYMOCYTOCHEMICAL STUDY OF ALKALINE PHOSPHATASE ISOENZYMES IN GASTRIC CANCER-LIGHT AND ELECTRON MICROSCOPIC OBSERVATIONS

By using light and electron microscopic cytochemical technique, the activities and distributions of AP isoenzymes in gastric cancers and benign gastric diseases were examined.The results showed: Nagao, Regan and Kasahara isoenzymes, which were not expressed in normal gastric mucosae and non-malignant lesions, might be considered as tumor markers of gastric cancer;The epithelium of intestinal metaplasia exhibited intestinal-type AP only,which was one of the markers of well-differentiated intestinal metaplasia.In the view-point of the gene expression of AP isoenzymes, two mutation hypothesis and recessive-gene mutation hypothesis might be fit for gastric cancer.

Effects of Several Doses of ^(60)Co Gamma Irradiation on Mouse Peritoneal Macrophages

Effects of several doses of60Co gamma irradiation on mouse macrophages were in-vestigated by means of quantitative cytochemistry.It was found that the activities of lysosomalenzymes（acid phosphatase（AcPase）and a-naphthyl acetate esterose（ANAE））,adenosinetriphosphatase（ATPase）and positive substances of periodic acid Schiff’s reaction（PAS）inperitoneal macrophages were increased after total-body irradiation （1,3Gy）,and decreasedwith the increase of doses of total-body irradiation（5,8Gy）.Meanwhile,the phagocytic ca-pacity and the size of peritoneal macrophages of irradiated mice also changed as compared withthe control group.

Stereological and cytochemical study on the pathogenesis of hepatitis induced by halothane in rats

This study was designed to investigate the relationship between hepatotoxicity of halothane and altered hepatic calcium homeostasis in enzyme-induced hypoxic rats by means of Ca2+ cytochemical location and EDX microanalysis combined with stereology of liver and ultrastructural analysis by computer. It was found that more calcium precipitated in the cytoplasm and mitochondria of liver than that in the controls following halothane exposure. This alteration appears to be the result of accelerated uptake of Ca2+ by hepatocytes and decreased output of loaded Ca2+ on the one hand, and increased release of reserved Ca2+ on the other hand. These results suggest that change in cullular Ca2+ balance plays an important role in the mechanism of hepatotoxicity induced by halothane exposure.

Cytochemical, Structural and Ultrastructural Characterization of Tetrasporogenesis in Bostrychia radicans (Ceramiales, Rhodophyta) from the Mangroves of Itacorubi and Rio Ratones, Santa Catarina, Brazil

Little is known about the morphology and location of macromolecules, especially proteins and carbohydrates, in vegetative and reproductive structures of mangrove species, including Bostrychia radicans. Therefore, to gain a better understanding of tetrasporogenesis in B. radicans, cytochemical, structural and ultrastructual analyses were performed. Thalli were collected from mangroves in Florianópolis, Santa Catarina, Brazil. Fertile branches were fixed and processed for light microscopy (LM), confocal microcopy and transmission electron microscopy (TEM) observations. The LM sections were stained with toluidine blue, periodic acid-Schiff and Coomassie brilliant blue. Tetrasporogenesis occurs in the pericentral cells of the terminal branches. This process is initially characterized by an increase in cell volume, resulting from the proliferation of organelles. The young tetrasporangia remain connected to the basal cell by pit connections. After a considerable increase in volume, the tetrasporocytes divide tetrahedrally, giving rise to haploid spores. During this process, there is an increasing production of starch grains, causing the organelles to group. As the organelles proliferate, the plasma membrane undergoes simultaneous invaginations toward the tetrasporangium center. The most conspicuous organelle throughout tetrasporogenesis was the Golgi complex. Polysaccharidic components are predominant in the tetrasporangium cell wall throughout tetrasporogenesis. Although protein components prevail in the cytoplasm of younger tetrasporangia, there is a predominance of reserve material with maturation. In the initial phase, there is an increase in the number of chloroplasts and a significant increment of Golgi bodies which contribute to the formation of the amorphous portion of the cell wall and possibly the biosynthesis of starch grains.

A Biphenotypic (Mixed Phenotypic) Acute Leukemia: Report of Two Cases with Immunophenotypic Study

Biphenotypic acute leukemia (BAL) is an uncommon clinical entity. It is a type of acute leukemia with features characteristic of both the myeloid and lymphoid lineages and for this reason is designated as mixed-lineage, hybrid or biphenotypic acute leukemia. As strict diagnostic criteria have only recently been established, the precise incidence among acute leukemia is uncertain, although it is likely to account for approximately less then 5% of all acute leukemia. BAL is now collectively considered as “mixed phenotype acute leukemia” (MPAL). We hereby report two cases of a rare disease, BAL from our institution in the light of morphology, cytochemistry, flow cytometry and review of literature regarding these cases are described.

A novel alkaline phosphatase positive granule in rat neutrophils

The localization of the alkaline phosphatase (ALPase) activity in isolated rat neutrophils was ultrastructurally observed to further study the function and mechanism of the neutrophils in defending the body against invading microbes. Methods: The rat neutrophils were isolated from the whole blood and peritoneum. The ALPase cytochemical and ultrastructural observation were performed in the research. Results: It was found that ALPase-positive granules were localized in both rat blood and peritoneal neutrophils,which were different from the traditional azurophil and the special granules, and located in cytoplasm in the forms of some novel vacuoles and slender rod-shaped structures. These structures were easily upregulated by phorbol 12-myristate 13-acetate (PMA ) and/or N-formyl--fen--phe (fMLP) treatment, and shifted toward the surface of the cells. The upregulation was easily observed 2. 5 min after the stimulation with PMA. Most of the alkaline phosphatase-positive granules fused with each other at first, and some of them were connected with the cell surface plasma membrane at 15 min after the stimulation with PMA or fMLP. Conclusion: A novel alkaline phosphatase - positive granule exists in isolated rat neutrophils and may originate from the Golgi complexes of the neutrophils. The activity of the granules relates to at least activation of protein kinase C.

Ultrastructural features of the hypopharyngeal glands in the social wasp Polistes versicolor （Hymenoptera： Vespidae）

The wasps of the genus Polistes have been considered the key to understanding the evolution of social behavior in Hymenoptera. Several studies have shown that the development of organized insect societies was accompanied by the evolution of structures like exocrine glands, which became specialized to perform specific functions. This article investigates the ultrastructural and cytochemical features of the hypopharyngeal glands of Polistes versicolor. These glands have been studied in depth in social bees, where they occur only in nurses and produce the royal jelly. Our results＇ revealed that these glands basically did not vary among individuals or between sexes. They are constituted by spherical cells, each with a large nucleus and well-developed rough endoplasmic reticulum. Secretion vesicles are abundant, but lipid droplets were not observed, indicating that these glands may not have a role in pheromone synthesis. Acid phosphatase was detected in lysosomes, and also flee in the cytosol, but did not seem to be related with cell death. Thus, our results suggest that the hypopharyngeal glands ofP. versicolor may not have a specialized social role, but could produce digestive enzymes.