Lysosomal origin of the chloragosomes in the chloragogenous tissue of the earthworm Eisenia foetida: cytochemical demonstration of acid phosphatase activity

Summary The cytochemical localization of the lysosomal marker enzyme acid phosphatase was studied in the chloragogenous tissue of earthworms. The Gomori lead technique and the cerium capture technique were utilized. Both techniques demonstrated the chloragosomal location of this enzyme. Only a small proportion of chloragosomes presented reactivity, which suggests that these organelles are distinctly heterogeneous. The reaction product was localized in the periphery of chloragosomes, suggesting a membrane-bound compartmentalization of acid phosphatase. In addition, degenerating mitochondria and membrane whorls were observed in some chloragosomes, indicating the possibility that these organelles perform autophagosomal functions.     

The effect of lead incorporation on the elemental composition of earthworm (Annelida,Oligochaeta) chloragosome granules

Summary The elemental compositions of chloragosome granules in the earthworm Lumbricus rubellus living in non-polluted (Dinas Powys) and heavily Pb-polluted (Wemyss) soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. High Pb concentrations were found in chloragosomes of Wemyss animals; Pb was not detected in chloragosomes of Dinas Powys animals. Partial correlation and regression analysis indicated that the in vivo accumulation of Pb by chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Pb is bound by P-containing ligand(s) in the chloragosome matrix. The sequestration of Pb by chloragosomes results in the detoxification of the metal by accumulative immobilization.     

The effect of lead incorporation on the elemental composition of earthworm (Annelida, Oligochaeta) chloragosome granules

The elemental compositions of chloragosome 'granules' in the earthworm Lumbricus rubellus living in non-polluted (Dinas Powys) and heavily Pb-polluted (Wemyss) soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. High Pb concentrations were found in chloragosomes of Wemyss animals; Pb was not detected in chloragosomes of Dinas Powys animals. Partial correlation and regression analysis indicated that the in vivo accumulation of Pb by chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Pb is bound by P-containing ligand(s) in the chloragosome matrix. The sequestration of Pb by chloragosomes results in the detoxification of the metal by accumulative immobilization.     

Zinc sequestration by earthworm (Annelida: Oligochaeta) chloragocytes. An in vivo investigation using fully quantitative electron probe X-ray micro-analysis

The elemental compositions of chloragosome "granules" in the earthworm Lumbricus rubellus living in non-polluted and heavily Zn-polluted soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. The in vivo accumulation of Zn by the chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Zn was apparently bound by at least two ligand pools (Pool 1 = uncharacterised; Pool 2 = P-containing ligands, binding approximately 45% and 55% of the Zn, respectively) in the "control" chloragosomes. In Zn-contaminated chloragosomes, most (approximately 70%) was bound by P-containing ligand(s) but some (less than 1%) was also bound by S-containing ligands. It is suggested that the sequestration of Zn in chloragosomes results in the detoxification of the metal by accumulative immobilisation.     

The elemental composition of the chloragosomes of two earthworm species (Lumbricus terrestris and Allolobophora longa) determined by electron probe X-ray microanalysis of freeze-dired cryosections

Summary The elemental composition of the chloragosomes of the two earthworm species, one with complex highly active Ca-secreting calciferous glands (L. terrestris) and the other with non-secretory glands (A. longa), was determined by the electron probe X-ray microanalysis of unfixed freeze-dried cryosections. The predominant constituents of the chloragosomes of both species were P, Ca, Zn and S, with lesser quantities of K, Cl and Fe. The most striking species differences in chloragosomal chemistry were the higher concentrations (expressed as relative mass fractions) of P(×2.1), Ca(×1.5), and Zn(×2.3) in L. terrestris, and the much higher S(×19.6) in A. longa. These differences were discussed in relation to the general ecophysiology of the two species, and more specifically in relation to heavy metal uptake and binding.     

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.     

Zinc sequestration by earthworm (Annelida: Oligochaeta) chloragocytes

Summary The elemental compositions of chloragosome granules in the earthworm Lumbricus rubellus living in non-polluted and heavily Zn-polluted soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. The in vivo accumulation of Zn by the chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Zn was apparently bound by at least two ligand pools (Pool 1=uncharacterised; Pool 2=P-containing ligands, binding approximately 45% and 55% of the Zn, respectively) in the control chloragosomes. In Zn-contaminated chloragosomes, most (70%) was bound by P-containing ligand(s) but some (<1%) was also bound by S-containing ligands. It is suggested that the sequestration of Zn in chloragosomes results in the detoxification of the metal by accumulative immobilisation.     

Subcellular localization of zinc in experimentally polluted tubifex tubifex (Annelida,Oligochaeta)

• 1.1. Localization of Zn (+ ⁶⁵Zn) has been examined within twelve subcellular fractions (derived from discontinuous sucrose gradients) of preincubated T. tubifex.
• 2.2. Zn was principally associated with the pellet (28% of total) and lowest density fraction (14%).
• 3.3. Pellet ultrastructure is composed of chloragosomes and epicuticle. Pellet Zn is localized within chloragosomes, X-ray microanalysis showing chloragosomal Zn concentration to exceed epiculticular Zn by a factor of thirty.
• 4.4. Biochemical and ultrastructural studies demonstrate that Zn is not appreciably bound to other cell constituents.
• 5.5. Chloragosomal localization of internalized Zn indicates a capacity for detoxification.

     

Malathion Toxicity: Effect on the Ovary of the Zebra Fish Brachydanio rerio (Cyprinidae)

The nucleic acids (DNA and RNA), protein contents and the activity of alkaline phosphatase was significantly decreased in the ovary of Brachydanio rerio after exposure to different concentrations of malathion, an organophosphorus pesticide. Total free amino acids and acid phosphatase activity, however, were enhanced. The decrease in the measured parameters depended on both concentration and time. Histopathological studies revealed developmental arrest at all levels from oogonium to tertiary stage oocytes. After transfer to fresh water without the pesticide, there was a marked trend of recovery in al' the parameters studied.     

Effect of endocytosis upon acid phosphatase activity ofTetrahymena pyriformis

Summary Increased endocytosis inTetrahymena pyriformis, produced by presenting starved cells with either peptone-yeast extract medium or killed yeast cell suspension, results in increased cellular acid phosphatase activity.Tetrahymena, grown in peptone-yeast extract medium, showed increased acid phosphatase activity after phagocytosis of yeast cells. This increase was not apparent until about one hour after presentation and was maximal at about 2.5 hours.Tetrahymena, grown on yeast suspension, showed little increase in acid phosphatase activity on presentation with peptone-yeast extract medium. These results may indicate that endocytosis, of either particles or solutes, produces an adaptive increase in acid phosphatase activity (presumably lysosomal in nature) which is related to feeding.Histochemical examination failed to localise the increase in acid phosphatase activity cellularly, but small particles, of about 1 diameter, which showed acid phosphatase activity and were presumably lysosomes were noted. Closely orientated yeast cells showed varying intensities of lead deposition, from absence to intense staining. This suggests that newly ingested yeast cells may be ingested initially in a single phagosome and that thereafter one or more lysosomes may fuse with them.     

Phase and Period Responses of the Two-Peak Circadian Rhythm of Trigonoscelis gigas Reitter (Coleoptera: Tenebrionidae) for 6-hr Light Pulses

Abstract

Enzymatic activity of five lysosomal hydrolases: acid p‐nitrophenyl phosphatase (EC 3.1.3.2), acid β‐glycerophosphatase (EC 3.1.3.2), arylsulphatase (EC 3.1.6.1), β‐galactosidase (EC 3.2.1.23) and β‐N‐acetylhexosaminidase (EC 3.2.1.30) was studied in the supernatants of homogenates of hearts of unirradiated mice, serving as controls, and a group of U.V.‐irradiated mice.

In the control group, determinations made at 6‐hr intervals showed rhythmic diurnal changes in activities of three acid hydrolases. These changes were statistically significant in the case of acid p‐nitrophenyl phosphatase, acid β‐glycerophosphatase, and β‐N‐acetylhexosaminidase. The effect of U.V.‐irradiation was manifested mainly by depression of enzyme activities of the acid hydrolases during the first few hours after exposure. Depression of activities of arylsulphatase and β‐N‐acetylhexosaminidase by U.V. was statistically significant. Presumably, the fall in enzyme activities of the acid hydrolases was due to chemical mediators formed in the skin under the influence of U.V.‐irradiation and adrenal corticoids secreted into the blood.     

Acid phosphatase activity during spore differentiation of the red algaeGigartina teedii andChondria tenuissima

The acid phosphatase activity during carposporogenesis inGigartina and tetrasporogenesis inChondria was studied using the Gomori technique. During the first steps of gonimoblast maturation ofGigartina, portions of cytoplasm are ensheathed by ER cisternae with acid phosphatase activity, giving rise to autolysosomal concentric membrane bodies. In a similar way large mucilage sacs are severed. They extrude their contents in a kind of exocytosis. Multivesicular bodies, concentrically arranged cisternae and extracytoplasmic compartments, each with acid phosphatase activity, remain in young carpospores for some time, probably as remnants of the autophagocytotic and exocytotic events. The Golgi apparatus is poorly developed in gonimoblast cells and young carpospores. It becomes a prominent cell component in maturing carpospores and then participates in cell wall formation. Only some of the dictyosomal cisternae contain acid phosphatase; these are irregularly distributed in the dictyosome. — In pre- and postmeiotic tetraspore mother cells ofChondria massive lead deposits are found in the dictyosomes and in adjacent Golgi vesicles. Finer lead precipitates occur in ER cisternae, especially in those which are sequestering starch-grain-containing portions of the cytoplasm to give rise to autolysosomes. During cell cleavage, the dictyosomes aggregate. They become devoid of acid phosphatase activity with the exception of vesicles at the trans face. Later, Golgi stacks associate and have common, Gomori positively reacting, narrow cisternae at the cis face. The Golgi apparatus derived cored vesicles do not contain lead precipitates whereas the Golgi cisternae in the final stage of tetrasporogenesis show acid phosphatase activity. Variations in acid phosphatase distribution are explained in the light of current models of membrane flow.Dedicated to Univ.-Prof. DrO. Härtel on the occasion of his 80th birthday.     

Effect of repeated intraperitoneal exposure to picrotoxin on rat liver lysosomal function

Effect of repeated (20 days) exposure to picrotoxin (PTX) on rat liver lysosomal function was evaluated by measuring the free and total activities of acid phosphatase, cathepsin D, ribonuclease II (RNAse II) and deoxyribonuclease II (DNAse II). The free activities of the nucleases (both RNAse II and DNAse II) were increased following PTX exposure. The total DNAse II activity was increased by 2.2-fold whereas the total acid phosphatase activity was decreased by 28%. Consequently, the ratios of total activity / free activity were low in the PTX exposed groups, implying loss of membrane integrity. Cathepsin D activity was completely abolished. The results show that repeated exposure to PTX can lead to lysosomal dysfunction in liver.     

Effect of control of pH on the excretion of acid phosphatase by Rhodotorula glutinis

Summary The excretion of an acid phosphatase by Rhodotorula glutinis is related to the pH of the medium. During growth, the phosphatase excretion into the medium at a constant pH of 4.5 was 5 times higher than that observed at variable pH. After cultivation at a constant pH of 4.5 or at variable pH, cells were incubated at various pH values between pH 2 and 7. During this second incubation acid phosphatase release occured at pH 4.5 to 6.5 only. There was no release at pH 3.0; but when resting cells incubated at this pH were placed in a buffer solution at pH 5.5 a high activity was released. Extensive washing did not eliminate residual intrinsic acid phosphatase activity. These two types of acid phosphatase were phosphomonoesterases with an identical specificity for different substrates.     

Studies of vegetative compatibility-incompatibility in higher plants

Summary In order to elucidate the events that lead to cellular autolysis, and thus better understand the mechanism of cellular incompatibility betweenSedum telephoides andSolanum pennellii stems, we have followed the appearance and fate of the hydrolytic enzyme acid phosphatase in both the compatibleSedum autograft and the incompatibleSedum/Solanum heterograft. Acid phosphatase was localized by a modified Gomori-type reaction. Following an initial association with the endoplasmic reticulum and dictyosomes by 6–10 hours after grafting, acid phosphatase activity in the compatibleSedum autograft was associated primarily with the plasmalemma, tonoplast, and vacuole. This strict compartmentation in membranes or organelles and absence of enzyme from the cytosol was maintained throughout the development of the compatible autograft inSedum. Although acid phosphatase activity in the incompatible heterograft betweenSedum andSolanum was initially similar to the compatible autograft inSedum, a marked difference in enzyme localization occurred in the two graft partners over time.Solanum cells accumulated increased amounts of acid phosphatase, but the enzyme remained sequestered in the plasmalemma, tonoplast, and vacuole. In comparableSedum cells, however, there was a dramatic increase in acid phosphatase activity in the cytosol, often without any prior compartmentation within the vacuole. This high activity of acid phosphatase in theSedum cytosol was correlated with cellular autolysis, death, and eventual cell collapse to form the characteristic necrotic layer that insulates the stock from the scion. These results suggest that the lethal cellular senescence associated withSedum cells of the incompatible heterograft is correlated with a cytoplasmic release of acid phosphatase. A similar release of the enzyme does not occur in theSolanum stock or in the compatibleSedum autograft. Thus, while acid phosphatase synthesis and/or activation is induced in both the compatible and incompatible grafts, incompatibility betweenSedum andSolanum involves a failure ofSedum cells to isolate hydrolytic enzymes from the cytosol, which subsequently leads to cellular necrosis.Supported in part by grants from the Academic Senate of UCLA, Sigma Xi, the American Philosophical Society, and the URC of Baylor University.     

Effect of Phosphorus Nutrition on the Cellular Distribution of Acid Phosphatase in the Leaves of Lycopersicon esculentum L.

A histochemical study using light microscopy has been made ofthe distribution of acid phosphatase (EC 3.1.3.2 [EC] ) activity intransverse sections of fully expanded leaves of Lycopersiconesculentum grown in phosphate-deficient or sufficient media.Leaf tissues were prepared by two methods and were embeddedin paraffin wax. The location of acid phosphatase activity inleaf sections was determined by trapping orthophosphate releasedfrom p-nitrophenyl phosphate with lead acetate and subsequentlyconverting the lead phosphate to optically dense lead sulphide.In leaf sections from control tissue lead sulphide depositswere larpely confined to the spongy mesophyll cells. Whereasthe staining of the palisade cells was limited and of a granularnature, the staining of the spongy mesophyll cells was heavierand coincident with the outline of the individual cells. Moreover,the minor veins were more heavily stained than the surroundingmesophyll cells. Sections of phosphorus-deficient tissues wereheavily stained in both the palisade and spongy mesophyll layersand heavy deposits of lead sulphide were present in the regionsof the minor veins. It is suggested that the enhanced acid phosphataseactivity of the mesophyll cells in fully expanded leaves couldbe involved in the remobilization of phosphate within phosphorus-deficientplants, or be part of a phosphate transporting system, concentratingthe intracellular phosphate from the limiting supply in thesolution bathing the mesophyll cells. Lycopersicon esculentum L., tomato, acid phosphatase, phosphorus nutrition     

Ultrastructural localisation of acid phosphatase,non-specific esterase and β-glucuronidase in the midgut epithelium of Tenebrio molitor,Schistocerca gregaria and Carausius morosus

Summary Acid phosphatase, non-specific esterase and -glucuronidase have been localised in the midgut epithelium of three species of insect using naphthol esters as substrates and triphenyl-p-amino-phenethyl lead as coupling salt. In all three species acid phosphatase and -glucuronidase appear to be confined to primary and secondary lysosomes. Non-specific esterase activity was demonstrated within membrane-enclosing bodies in all three species, associated with lipid droplets in T. molitor and C. morosus and with an unidentified intranuclear structure in C. morosus.     

Cytochemical localization of acid phosphatases in the dimorphic fungusSporothrix schenckii

A modified Gomori procedure at the electron microscopic level revealed a multiplicity of acid phosphatase activity sites in both yeast-like and mycelial phase cells. Vacuoles and the periplasmic space contained electron opaque deposits (lead phosphate) that were absent in control incubations either lacking the substrate (-glycerophosphate) or fortified with an inhibitor (sodium fluoride). The outermost region of the cell envelope was also active and, in contradistinction to previous examples with other yeasts, deposition of lead phosphate in this locale occurred even when the rate of orthophosphate generation was drastically reduced by lowering the substrate concentration. When mechanically disrupted yeast-like cells were washed and then subjected to the cytochemical procedure, pieces of broken cell envelope gave a positive reaction. The reaction product was invariably restricted to one side of cell wall cross sections. A specific and novel association of acid phosphatase with a microfibrillar zone was indicated.     

Structural basis of cation exchange, complex formation and redox properties in chloragosomes.

Components of chloragosomes isolated from Octolasium transpadanum ROSA were separated by fractional extraction. The chemical composition of the fractions was determined and their effect on the anion- and cation-binding capability and on the lipophilic property of chloragosomes was studied. The acid-alcoholic extract of chloragosomes contained, among others, metalloporphyrins with 2 or 4 free -COOH groups; the residuum consisted of basic proteins. The metalloporphyrins and phosphatides gained by extraction with chloroform-methanol neutralize the basic groups of the chloragosome proteins, thus, an excess of free acidic groups develops, which is responsible for the polyanionic nature and cation exchange of chloragosomes. The apolar groups of phosphatides and carotenoids are responsible for the lipophilic nature of chloragosomes. Under experimental circumstances both in vivo and in vitro, organic cations were bound to chloragosomes, partially by a cation-exchange mechanism and partially by formation of more stable complexes. A small amount of anions may be absorbed by the bound cations. The diluted acidic extract of chloragosomes predominantly contain organic cations, in the present study riboflavin, flavin nucle0tide and thiamine were determined. Chloragosomes, owing to their structure, form complexes of varying stability with anions, cations and hydrophilic compounds equally. This feature explains their capability of cumulating trophic and toxic substances, while their redox activity is explained by their metalloporphyrin, flavin, thiamine and carotene content.     

Amelioration of beryllium induced alterations in hepatorenal biochemistry and ultramorphology by co-administration of tiferron and adjuvants

Influence of adjuvants i.e., α-tocopherol (25 mg/kg, p.o.) and piperine (10 mg/kg, p.o.) on therapeutic potential of chelator tiferron (300 mg/kg, i.p.) was evaluated to encounter toxicogenic events of beryllium exposure. Albino rats were exposed to beryllium nitrate (1 mg/kg, i.p.) daily for 28 days followed by treatment of aforesaid therapeutic agents for 5 consecutive days. Results were considered to be significant at p ≤0.01 and p ≤0.05. Exposure to beryllium increased its concentration in liver, kidney and serum causing significant alterations in the activity of CYP-450 2E1 system, microsomal lipid peroxidation and protein; alkaline phosphtase, lactate dehydrogenase, γ-glutamyl transpeptidase, bilirubin, creatinine and urea in serum; activity of acid phosphatase, alkaline phosphatase, adenosine triphosphatase, glucose-6-phosphatase and succinic dehydrogenase in liver and kidney. Beryllium exposure also induced severe alterations in histopathology and ultramorphology of liver and kidney proving its toxic consequences at cellular level. Tiferron along with adjuvants dramatically reversed alterations of all variables more towards control rather than individual treatment. Study concluded that tiferron in combination with α-tocopherol and piperine respectively was beneficial in diluting beryllium induced systemic toxicity; however, combination of tiferron and piperine presented more pronounced therapeutic potential.