Simultaneous localization of 3H-thymidine incorporation and acid phosphatase activity in mouse spleen: EM radioautography and cytochemistry.

Simultaneous localization of 3H-thymidine incorporation and acid phosphatase (AcP) activity was undertaken by combined radioautography and cytochemistry in the spleen of mice at different ages. The localization of radiolabelled thymidine was used to determine the site of DNA synthesis (cell proliferation), while AcP activity as a marker for cell lysis/death. For EM radioautography (EMRAG), the tissue sections were incubated in a medium containing 3H-thymidine and processed for radioautography, while the lanthanide-based method for the ultrastructural localization of AcP activity was employed. Quantitation of AcP activity was carried out by X-ray microanalysis. In all tissue sections examined, mostly of the labelled nuclei were observed in the hematopoietic cells. Few mitochondria of these cells were labelled. The labeling index was expressed as the percentage of labelled cells over the total number of counted cells. The labeling indices dropped considerably from day one after birth and progressively until the 10th month. The result of AcP activity correlated well with the result of a previous work (Olea, 1991). The localization of radiolabelled thymidine and AcP activity were not hindered by the simultaneous exposure of the same tissue section to 3H-thymidine and AcP cytochemical media. Interestingly enough, the spleen actively participates both in hematopoiesis and erythrophagocytosis. Prominently, it is most active during the early postnatal life. However, their influence declined considerably at the later stage of life (adult stage).     

Acute and sub-acute effects of 2-butenoic acid-3-(diethoxy phosphinothioyl) methyl ester (RPR-II) on testis of albino rat

Acute and sub-acute toxic effects of a novel phosphorothionate coded as RPR-II on testis of albino rats were studied. In acute study rats received a single dose of 12.3 mg/kg of RPR-II and sacrificed after 24 hr. For sub-acute study 0.58 mg/kg/day was administered orally to rats for 10 and 21 days. Acute exposure of rats to RPR-II brought no change either in the gonadosomatic index (GSI) or in the structure of testis or in the serum levels of testosterone. Testis glutathione (GSH) level and glutathione S-transferase (GST) activity was significantly decreased whereas, acid phosphatase (AcP) levels increased significantly at 24 hr post-treatment. On 7th day (withdrawal period) after the cessation of the treatment the GSH, GST, AcP, and AkP levels reached to near control. The sub-acute study revealed a significant decrease in GSI on 10th and 21st day of the treatment. In contrast, a time-dependent and significant increased in GSH level and GST activity was observed on 100th and 21st day of post-treatment, except GSH level on 10th day, which was declined. Due to RPR-II treatment the testis AcP and alkaline phosphatase (AkP) levels were significant at both 10th and 21st day of medication but AcP levels were increased whereas AkP levels decreased. The histopathological studies on day 10th showed considerable loss of spermatozoids in testis and at 21st day complete derangement of cellular organization was observed. Testosterone levels decreased significantly on 10th day and remained significantly low at 21st day. However, withdrawal studies showed a recovery in testis of rat treated with RPR-II. GST, GSH, GSI, AcP and AkP values recovered, testosterone levels were also well recovered but recovery in testis structure remained at a low profile. The present study suggests that RPR-II may cause testicular toxicity in rats affecting the normal functioning of testis and it also gave some new information in withdrawal studies.     

Protein synthesis in the livers of aging mice studied by electron microscopic radioautography.

The application of 3H-leucine results in labeling of the liver cells of mice in which protein is synthesized at various ages of the animals. Quantitative changes of protein synthesis in the hepatocytes of aging mice were studied by electron microscopic radioautography. The silver grains in the hepatocytes were mainly located over the rough surfaced endoplasmic reticulum, mitochondria, Golgi apparatus, cytoplasmic matrix, and a few over the nuclei. The number of silver grains in the cytoplasm and nuclei of the hepatocytes gradually increased after birth, reached the maximum at 1 month after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the hepatocyte cytoplasm was more than that in nuclei at various ages. The number of silver grains in the rough surfaced endoplasmic reticulum and mitochondria gradually increased from embryo to 1 month after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the Golgi apparatus showed almost no change from fetal stage to 6 months after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the cytoplasmic matrix gradually increased from fetal stage to 2 months after birth, then decreased with aging until the 24th month. These changes reflect the quantity of protein synthesized in each cell organelle at various ages of animals.     

Number, activity and thermostability of the electrophoretic forms of acid phosphatase in Amoeba proteus, cultured at different temperatures

In free-living amoebae (Amoeba proteus, strain B), cultured at 10 and 25 degrees C, we compared the number, activity, and thermostability of separate electromorphs of Triton-soluble acid phosphatase (AcP) revealed by disc-electrophoresis in polyacrylamide gel using 2-naphthyl phosphate (pH 4.0) as a substrate. No differences in the number of AcP electromorphs and their mobility were observed at both these temperatures. The total activity of AcP electromorphas per unit of cellular protein and their total thermostability were lower in amoebae acclimated to 10 degrees C than to 25 degrees C. The above decrease may be a consequence of a simultaneous decrease in the activity and thermostability of two tartrate-sensitive electromorphs, both being of lysosomal nature. The total activity and thermostability of tartrate-resistant AcP electromorphs did not differ in amoebae acclimated to the two above temperatures. In amoebae cultured at 10 degrees C the fall of activity and thermostability of lysosomal AcP correlates with the decrease in their primary cell thermoresistance and phagocytic activity. The obtained results confirm the earlier conclusion (Vysotskaya et al., 1994) that lysosomes may be involved in acclimation of electrothermal animals to changing environmental temperatures.     

Interplay between acid phosphatase and cysteine proteases in mediating vitellin degradation during early embryogenesis of Periplaneta americana

In this work, we characterized the activities of two classes of proteases and AcP during early embryogenesis of Periplaneta americana. AcP activity was first detected at day 6 and reached a maximum level at day 10 of development. Using phosphoamino acids, phosphatase activity was shown to be directed only against phosphotyrosine at day 6 while at day 10 it was also active against phosphoserine. In parallel, two classes of proteases were detected and located within yolk granules: a clan CA-cysteine protease, which was inhibited by E-64, insensitive to CA 074 and activated by acidic pH at day 3; and a neutral serine protease, which was inhibited by aprotinin at day 6. Assays of vitellin (Vt) degradation evidenced that incubations at neutral pH induced slight proteolysis, while the incubations at acidic pH did not result in Vt degradation. However, pre-incubations of Vt with AcP increased the levels of Vt acidic proteolysis and this could be inhibited by the addition of phosphatase inhibitors. On the other hand, the same pre-incubations showed no effects on the profile of degradation at neutral pH. We propose that AcP and cysteine protease cooperate to assure Vt breakdown during early embryogenesis of P. americana.     

鸡胚巨噬细胞AcP酶变化及AcP酶与凋亡细胞的关系

本研究采用电镜及酶细胞化学的方法观察了鸡胚脾脏不同胚龄组巨噬细胞溶酶体酸性磷酸酶（AcP酶）的变化、凋亡实验组巨噬细胞及其AcP酶与凋亡细胞的关系。取10天、13天和17天鸡胚脾脏,按Gomori法显示AcP酶,各胚龄脾脏巨噬细胞AcP酶细胞化学反应阳性,按AcP酶染色阳性做溶酶体计数,结果显示随着胚龄的增加溶酶体数随之增加,尤以第17天组溶酶体数增加最为明显,所得数据经统计分析表明各胚龄组间溶酶体数的差异有统计学意义。凋亡实验组采用放线菌酮诱导15天鸡胚脾脏细胞凋亡,结果显示凋亡细胞为各类幼稚血细胞,以幼稚淋巴细胞为主。巨噬细胞未见凋亡,而是吞噬了大量的凋亡细胞和凋亡小体,AcP酶反应颗粒不仅出现在巨噬细胞的溶酶体、吞噬体,还见于高尔基复合体、内质网等。细胞AcP酶反应强度数字化结果表明：凋亡组酶活性显著高于对照组,差别有统计学意义,提示胚胎巨噬细胞在凋亡细胞出现时AcP酶活性增强,说明巨噬细胞吞噬和消化凋亡细胞或凋亡小体是通过AcP酶等活性物质来实现的。     

Maturation and degeneration of the fat body in the Drosophila larva and pupa as revealed by morphometric analysis

Using morphometric and cytochemical techniques we have described changes taking place in the fat body cells during three different stages of development. The cell number remains constant at about 2200 cells during larval life and then decreases gradually and continuously throughout metamorphosis and the first 3 days of the adult stage until no more cells can be observed. Cell size increases rapidly during the larval period and decreases steadily during metamorphosis and adult stage. The size of the nuclei increases during the larval instars and decreases during the pupal interval. The change in nuclear size is correlated with the amount of DNA present throughout development implying the nuclear DNA is synthesized during the larval period and degraded gradually during metamorphosis. The cell size changes are due in large part to accumulation or loss of reserve substances: lipid droplets, glycogen deposits and protein granules. During metamorphosis the amount of lipid decreases slightly whereas glycogen experiences two loss cycles. The protein granules in the form of lysosomes continue to increase in amount during the first day of metamorphosis because of a short period of massive autophagy. Then the lysosomes decrease in amount throughout the remainder of metamorphosis. The lysosomes stain positively for lipofuscin.     

Quantitative X-ray microanalysis of calcium with the Camebax-TEM system in frozen, freeze-substituted and resin-embedded tissue sections. Application to molluscan glio-interstitial granules

The relevance of the continuum method for a quantitative X-ray microanalysis of epon embedded tissue sections in the particular conditions offered by the Camebax-TEM system was tested and an improved model of specimen holder is proposed. The absolute calcium concentration [Ca] of membrane-bound intracellular glio-interstitial granules was determined by X-ray microanalysis in transmission electron microscopy of Mytilus retractor muscle. The Ca peak and background values were measured by the wavelength-dispersive spectrometer of the Camebax; the mass thickness of the section was recorded simultaneously with an added energy-dispersive detector. The tissue was frozen at approximately equal to 77 K in a mixture of liquid propane and butane, freeze-substituted in the presence of oxalic acid and embedded in epoxy resin. The calcium concentration of glio-interstitial granules can be as high as 180 mmol.kg-1 of epoxy-embedded tissue, with an average of 40 mmol.kg-1. The sampling of the data through repeated experiments is discussed and it is proposed that the cell would be the main level of variation. The Ca content of glio-interstitial granules is significantly lower in the tissues of animals submitted to high-potassium artificial sea-water for 10 min. This finding was predicted by the hypothesis that glio-interstitial tissue is a regulator of calcium concentration in extracellular spaces.     

Radiation-induced histochemical and ultrastructural changes in the enterocytes in the rabbit.

Ultrastructural changes and enzyme activities in the cell organelles of rabbits were studied within 1, 3, 6, 9, 15 and 30 days of the irradiation with 550 rads of gamma-radiation. Between the 1st and 3rd day after irradiation there was a fall in the succinate dehydrogenase activity in the swollen and frequently tigroidal mitochondria whose number distinctly diminished. By the 15th day these changes disappeared. In the hyaloplasm there was weakening of the reaction for lactate dehydrogenase after 1 day, and an increase in number of polysomes, glycogen granules and smooth vesicles after 3-9 days after irradiation. The glucose-6-phosphatase activity was unchanged and so was the shape of the Golgi apparatus. The activity of lysosomal enzymes and the number of lysosomes in the experimental groups was approximately normal. By the 6th day the activity of alkaline phosphatase in the striated border was lowered, subsequently normal. On the whole, the intensity of postradiation changes in the intestinal mucosal epithelium is correlated with the rhythm of proliferation and shedding of epithelial cells, although some signs of injury persist for longer time periods.     

X-ray microanalysis of cerium in mouse spleen cells demonstrating acid phosphatase activity using high voltage electron microscope

An X-ray microanalytical study was carried out on mouse spleen cells demonstrating acid phosphatase (AcP-A) activity, using cerium (Ce) as the capture agent at different accelerating voltages. The enzyme reaction products were localized in the lysosomes and appeared dense and homogeneous. The presence of cerium was confirmed by X-ray microanalysis. The main spectral line of cerium was present at La = 4.84 keV. The result showed that the X-ray count of Ce and the background (B) decreased significantly with increasing accelerating voltage between 100 and 400 kV. The change was more pronounced between 100 and 200 kV and thereafter, minimal change was noted. Consequently, the computed P/B ratio increased appreciably with increasing accelerating voltage. Thus, significant P/B ratio in X-ray microanalysis of biological specimens could be achieved by using a medium voltage transmission analytical electron microscope at accelerating voltage between 300 and 400 kV.     

Cold-stress induced formation of calcium and phosphorous rich chloragocyte granules (chloragosomes) in the earthworm Eisenia fetida

The cytochemical and functional characteristics of chloragocytes of both 'control' and cold-stressed Eisenia fetida were examined. Flow cytometry revealed the heterogeneity of chloragocytes: the first group was characterized by low, the second one by high acid phosphatase (AcP) content. In 'control' animals the former, in cold-stressed ones the latter type were the dominant form. The elevated AcP-activity correlated with the accumulation of autophagic vacuoles (AVs) in chloragocytes. Both AVs and all small chloragosomes showed high AcP activity, while most of the large chloragosomes did not display any. Most 'control' granules (0.75-1.25 μm) contained high amounts of Ca and P, with less and variable quantities of S, Cl, K, Fe and Zn. Small chloragosomes with low Ca and P concentrations were seldom found. In cold-stressed animals the number of small granules (0.25-0.75 μm) increased up to 40% of total population. Their Ca and P contents were significantly lower; S and Fe concentrations were higher than those of large chloragosomes (1.0-1.5 μm). Our results prove that the formation and elemental composition of chloragosomes can be influenced by environmental stressors and suggest that the mature chloragosomes are tertiary lysosomes and their formation is coupled to autophagocytosis.     

The effects of continuous illumination on the function and structure of Micrasterias torreyi Bail. (Conjugatophyceae)

Abstract. The division rate of Micrasterias torreyi cells grown under continuous illumination first accelerated but soon slowed down, and the cells lost their ability to divide after about 1 month. During the treatment the cells became pale green, the pyrenoids became fewer in number and defects appeared in the chloroplasts. After 1 month, the cells also soon died, even when subjected to intermittent illumination. The most striking structural alterations were found in the chloroplasts: the starch granules lost their typical structure, the lamellae were damaged and numerous electron dense precipitates appeared in the chloroplasts. The precipitates were similar to those formed in cells treated with supraoptimal external calcium concentrations and X-ray microanalysis showed that the precipitates were rich in calcium in both cases. The results suggest that light controls and activates the Ca²⁺ uptake in the plasma membrane as well as in the chloroplast envelope, that the large sized chloroplasts of Micrasterias are effective in regulation of cytoplasmic Ca²⁺ concentration, and that the injuries caused by continuous illumination may be largely due to the accumulation of Ca²⁺ in the chloroplasts.     

Cellular cadmium distribution in the common winkle, Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry

Various tissues of common winkles, Littorina littorea (L.), experimentally exposed to cadmium (Cd) chloride were examined using light and electron microscopy and their elemental composition determined by X-ray microanalysis and histochemistry. Membrane granules in gill epithelial cells with paddle cilia contain carbonates, phosphates and sulphides associated with different cations in different types of granules. Traces of Cd have been found only in those granules containing sulphur and iron. Nephrocytes also contain small amounts of this metal in the cytoplasm of excretory cells. X-ray microanalysis reveals that concretions of basophilic cells are minor sites for Cd sequestration while BTAN-ASSG stain for unbound Cd indicates that most of the Cd is located within the lysosomes of digestive cells in association with proteins. Low amounts of the metal have been evidenced in the granules of epithelial mantle cells rich in sulphur. The results also indicate that hemocytes contain granules of calcium phosphate and iron sulphide. Cd is also associated to sulphur rather than to phosphate. These hemocytes may act as Cd carrier from gills to kidney and digestive gland. A hypothetical pathway for Cd accumulation and detoxification is suggested.     

Acid phosphatases in the haemolymph of the desert locust, Schistocerca gregaria, infected with the entomopathogenic fungus Metarhizium anisopliae

A comparison has been made between the effects of wounding, chemical stimulation of the immune system and fungal infection on acid phosphatase (AcP) activity in the haemolymph of the desert locust, Schistocerca gregaria. Untreated control locusts had constitutive levels of AcP. As a lysosomal enzyme, AcP may have a role in autophagy and cell turn over as well as defence. Injection of saline and beta-1,3-glucan caused significant increases in haemocyte and plasma AcP. AcP activity also increased in the haemolymph on the 3rd day after inoculation with the entomopathogenic fungus M. anisopliae var acridum. This coincided with a decline in the total haemocyte count and a marked reduction in the proportion of plasmatocytes and coagulocytes that stained positive for AcP. Therefore a priori it seemed unlikely that the extra AcP in infected insects came from the host. A fungal origin for the enzyme was suggested by the identification of AcP isoforms from haemolymph of different treatments. Control inoculated (oil only) insects had an AcP at a pI of 4.3 that was stimulated further by the injection of laminarin. Additional isoforms appeared at around 7.3-7.5 in the laminarin treatment. However, the 4.3 isoform appeared to be suppressed in the insects infected with M. anisopliae var acridum. The band intensity was more like that of the control than the laminarin-injected insects. Two new isoforms appeared later on in infection. These enzymes had pIs that corresponded to some of the AcPs produced in vitro by the fungus. The results are discussed in the light of the possible benefits of secreted fungal acid phosphatases to the pathogen.     

Cellular cadmium distribution in the common winkle,Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry

Summary Various tissues of common winkles,Littorina littorea (L.), experimentally exposed to cadmium (Cd) chloride were examined using light and electron microscopy and their elemental composition determined by X-ray microanalysis and histochemistry. Membrane granules in gill epithelial cells with paddle cilia contain carbonates, phosphates and sulphides associated with different cations in different types of granules. Traces of Cd have been found only in those granules containing sulphur and iron. Nephrocytes also contain small amounts of this metal in the cytoplasm of excretory cells. X-ray microanalysis reveals that concretions of basophilic cells are minor sites for Cd sequestration while BTAN-ASSG stain for unbound Cd indicates that most of the Cd is located within the lysosomes of digestive cells in association with proteins. Low amounts of the metal have been evidenced in the granules of epithelial mantle cells rich in sulphur. The results also indicate that hemocytes contain granules of calcium phosphate and iron sulphide. Cd is also associated to sulphur rather than to phosphate. These hemocytes may act as Cd carrier from gills to kidney and digestive gland. A hypothetical pathway for Cd accumulation and detoxification is suggested.     

A method for the ultrastructural demonstration of non-specific esterase in human blood and lymphoid tissue

Summary Using the substrate 2-naphthylthiol acetate (NTA), we developed a reproducible method of demonstrating a non-specific esterase while retaining nuclear and cytoplasmic details at the ultrastructural level. The NTA esterase had a distribution and pattern of staining similar to those of esterases demonstrable at the light microscopic level by the -naphthyl acetate or naphthol AS-D acetate esterase reaction.The NTA esterase appeared as intensely electron-dense granules of varying size and shape in the cytoplasm. The granules were most abundant in the cells of the histiomonocytic series. The large number of diffusely scattered granules in the cytoplasm of the histiocytes and monocytes made it possible to separate these cells from other haematopoietic elements. There was usually no direct relationship between the NTA esterase positivity and the amount or the location of lysosomes or mitochondria, although in some histoocytes the granules appeared to the associated with lysosomes. The NTA esterase-positive granules were usually more numerous than lysosomes and were located outside the lysosomal granules. Some of the lymphocytes outside the germinal centres and most of the lymphocytes in the blood showed a punctate positivity in the form of 1–4 electron-dense dots. Plasma cells were usually negative but, in rare cases, contained an occasional single dot-like reaction product similar to that in some of the lymphocytes. Granulocytes were always negative.The method described in this paper can be used effectively for identification and study of human haematopoietic cell lines at the ultrastructural level.     

Estimation of the copper-iron correlation in the liver under experimental conditions. A microscopic and microchemical study

Liver obtained from copper-intoxicated rats was studied by electron microscopy and electron probe microanalysis. Large ultrastructural disorders in liver cells appeared at the level of lysosomes and mitochondria. Diffuse and aggregate copper granules were observed in the entire cell, but mainly in pericentrolobular and periportal zones. Probe analysis revealed an increased copper deposit, particularly during the chronic copper intoxication, and a very high concentration of iron gradually accumulated.     

Structural features of the submandibular salivary gland of gnotobiotic rats

The submandibular salivary gland was histochemically and electron microscopically studied in gnotobiotic and conventional Wistar rats at the age of 15 days--10 months. By the first month of age, the submandibular salivary glands in both groups of animals complete differentiation of their acini, and the gland, according to the type of its secretion, becomes seromucous with predominance of albuminous component in it. Succinate dehydrogenase activity and nonspecific esterase predominate in epithelium of the excretory ducts. At the second month of life some signs of moreasing ductal part of the gland appear in the gnotobiotic rats. This part greatly increases by the 4th--10th month of life, mainly at the expense of twisted granular sections. This increase in number and volume of the twisted granular ducts results in compressing acini and in ultrastructural disorders of granulocytes, with destruction of some mitochondria, increasing number of lysosomes, decreasing size and alterations in character of secretory granules, in structural disorders of the laminar complex. All these manifest depressing secretory function of granulocytes in the gland of gnotobiotic animals.     

Precursors of monoamine-storage organelles in developing megakaryocytes of the rat

Summary Identification and distribution of the precursors of aminestorage organelles in rat megakaryocytes during cell maturation were studied, using the uranaffin reaction for adenine nucleotide. The precursors of the amine-storage organelles appeared as 200–300 nm vesicles having an uranaffin electron dense granule, whereas they appeared as empty vesicles by conventional glutaraldehyde-OsO₄ fixation. X-ray probe microanalysis confirmed the existence of U and P in the uranaffin reaction positive vesicles. The precursors appeared in the immature megakaryocytes, especially at the trans(mature) face of the Golgi apparatus, and rapidly increased in number in the maturing cells. The size of the uranaffin granules in the precursory organelles increased gradually during cell maturation and became almost equivalent to the dense body of blood platelets in the final stage of cell maturation.     

Histochemical,autoradiographic and electron microscopic investigations of the renal proximal tubule of male and female rats after castration

Summary After castration of 90-day-old male and female rats, changes appear in the renal proximal tubule. A distinction can be made between early changes (up to 10th postoperative day) and later changes (20th–30th postoperative day). Between the 3rd and 5th day after castration the kidney of the females shows an increase in free estrogen receptors (biochemical studies) which are localized in the pars contorta of the proximal tubule (autoradiographic studies), while the male kidney shows a marked increase in urinary protein excretion up to the 10th day after castration. Proximal tubule changes detectable histochemically and electron microscopically do not appear until day 20 or 30 after castration. The results of castration are similar in segments S₁ and S₂. By days 20 and 30 after castration there is a decrease in the activity of lysosomal enzymes (acid phosphatase, acid -galactosidase). Electron microscopy shows a conspicuous decrease in the number of giant lysosomes (mainly in females) and apical vacuoles (mainly in males). A marked increase in the number of lysosomes is found in the S₃ segment; females always have more lysosomes than males. The number of peroxisomes is also greatly increased; they appear circular in the females but can assume bizarre shapes in the males. Lipid droplets appear in the basal region of the tubule cell of segments S₂ and S₃ in the males. The sex differences are preserved in all segments even after castration and become even more pronounced in the S₃ segment.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)Dedicated to Prof. Dr. W. Graumann, Department of Anatomy, University of Tübingen, on the occasion of his 65th birthday