Acid-phosphatase activity in sieve-tube members of Tilia americana

Summary Acid phosphatases are localized on the internal strands of sieve-tube members in the secondary phloem of Tilia americana. Companion cells and certain strand parenchyma cells associated with sieve-tube members show a high activity of acid phosphatases.This research has been supported by NSF Grant GB-615.     

Activation of β-Glucan Synthases by Wall-Bound Purple Acid Phosphatase in Tobacco Cells

Wall-bound purple acid phosphatases have been shown to be potentially involved in the regulation of plant cell growth. The aim of this work was to further investigate the function of one of these phosphatases in tobacco (Nicotiana tabacum), NtPAP12, using transgenic cells overexpressing the enzyme. The transgenic cells exhibited a higher level of phosphatase activity in their walls. The corresponding protoplasts regenerating a cell wall exhibited a higher rate of β-glucan synthesis and cellulose deposition was increased in the walls of the transgenic cells. A higher level of plasma membrane glucan synthase activities was also measured in detergent extracts of membrane fractions from the transgenic line, while no activation of Golgi-bound glycan synthases was detected. Enzymatic hydrolysis and methylation analysis were performed on the products synthesized in vitro by the plasma membrane enzymes from the wild-type and transgenic lines extracted with digitonin and incubated with radioactive UDP-glucose. The data showed that the glucans consisted of callose and cellulose and that the amount of each glucan synthesized by the enzyme preparation from the transgenic cells was significantly higher than in the case of the wild-type cells. The demonstration that callose and cellulose synthases are activated in cells overexpressing the wall-bound phosphatase NtPAP12 suggests a regulation of these carbohydrate synthases by a phosphorylation/dephosphorylation process, as well as a role of wall-bound phosphatases in the regulation of cell wall biosynthesis.     

Histophysiological studies on the corpus allatum of Leucophaea maderae

Summary On the basis of the occurrence, at the light microscopic level, of alkaline and acid phosphatases, the pigment epithelium covering the posterior surface of the iris in the albino rabbit can be divided into two zones not previously described, viz. a central zone close to the pupil, approximately corresponding to the area occupied by the iridic sphincter muscle, and a peripheral zone extending to the ciliary body. The central zone which is in intimate relation with the lens was found to have a high content of both phosphatases. At the fine structural level it exhibits a marked pinocytotic activity in the epithelium at the interdigitations between adjacent cells. Electron microscopy revealed that acid phosphatase is localized to the walls of the pinocytotic vesicles. Alkaline phosphatase is in evidence at the surface membrane folds and at microvillous processes between the epithelial cells and the adjoining muscle cells. Unlike the distribution of the acid phosphatase, that of the alkaline phosphatase does not differ fundamentally in the two zones at the fine structural level.In a series of dehydrogenases studied, staining with a view to succinic-, isocitric- and glucose-6-phosphate dehydrogenases revealed an evenly distributed content of enzyme throughout the epithelium. As to the lactic- and -hydroxybutyric dehydrogenases, contents seem to be lower in the pupillary than in the peripheral zone.     

Effect of Cycloheximide on the Encystment and Ultrastructure of the Ciliate, Histriculus

SYNOPSIS The effect of cycloheximide on the experimentally induced synchronous encystment of a hypotrich ciliate, Histriculus , was examined and the cytoplasmic ultrastructures of the control and the inhibitor-treated organisms were compared. Encystment was divided into the following 5 stages based on observations of living cells: stage 1, cells with brown cytoplasm; stage 2, cells with smooth, transparent cytoplasm: stage 3, spindle-shaped cells; stage 4, spherical cells without cyst walls; and stage 5, young cysts with cyst walls. When cycloheximide treatment was preceded by stage 2, encystment and transformation into the next stages were totally blocked, whereas even in the presence of the inhibitor, stage 4 and stage 5 cells encysted normally, and stage 3 cells transformed into stage 4 although they did not form cyst walls. On the basis of ultrastructural studies it is suggested that the formation and excretion of ectocyst precursors are severely inhibited by cycloheximide and that polysome formation, active in stages 1 and 2, might be almost finished by stage 3. The role of lysosomal enzymes in the early stages of encystment are discussed.     

Effects of UV-Radiation on Kinetics of Encystment and on Morphology of Resting Cysts of the Ciliate Laurentiella acuminata1

The effects of ultraviolet radiation (λ= 254 nm) on the kinetics of encystment of the hypotrichous ciliate Laurentiella acuminata and the structure of resting cysts obtained from irradiated precystic cells are reported. High doses of UV-radiation caused a delay of encystment with a linear increase in the average time for obtaining 50% of encystment (EN₅₀). Resting cysts with abnormal cyst walls were obtained when precystic cells were irradiated in the exposure range 720 to 960 J/m². The cystic layer (mesocyst) was approximately twice as thick (6.5 μ m) as normal (3.7 μ m). Microscopical observations of abnormal cysts revealed the presence of two complete mesocysts, and the absence of the spines characteristic of the ectocyst. The UV-dependent effects on the cyst wall were gradually corrected in successive generations of the irradiated cells.     

Structure and cytochemistry of the procambium in Salix buds during dormancy and dormancy breaking

This report presents a combined investigation of ultrastructural and enzymatic changes in the procambium from late winter to early spring. In January the procambial cells of dormant Salix buds have a convoluted plasma membrane with many plasmalemmasomes, numerous lipid bodies, large stacks of rough ER and plastids surrounded by smooth ER profiles. Several small lysosomes show activity of ATPase and acid phosphatases. In addition ER, nuclear envelopes, dictyosomes, and thylakoids have ATPase activity, and ER and plasmalemma, and nuclei also show acid phosphatase activity. In February metabolism seems to increase as indicated by lysosomes with membranous formations, dilated ER, nuclear envelopes, spiny vesicles, and polysomes. ATPase activity occurs in plasmalemma and vacuoles, and acid phosphatases in the middle lamella region of walls, in plasmalemma, vacuoles, ER, and nuclei. At the end of March, when growth starts inside the buds, but before they break, the stacks of rough ER disappear, and the vacuoles coalesce. Most of the lipid bodies have disappeared and the plastids have accumulated starch. Cell division and differentiation of procambial cells to protophloem and protoxylem have started. The distribution of ATPase increases; activity is found in walls and plasmalemma, and only a few small vacuoles still have ATPase and acid phosphatase activity. Notable is the appearance of ATPase in mitochondrial cristae and nucleoli and the occurrence of rather high levels also in endomembranes and dictyosomes.     

Localization of chitin on ultrathin sections of cysts of two ciliated protozoa,Blepharisma undulans andPseudomicrothorax dubius,using colloidal gold conjugated wheat germ agglutinin

Summary The cyst walls of the ciliatesBlepharisma undulans andPseudomicrothorax dubius were examined ultrastructurally and by postembedding labeling with wheat germ agglutinin (WGA)-gold conjugate. Different methods of fixation and embedding were performed. In all procedures, WGA-gold binds selectively to material of the cyst wall. Pretreatment of the sections with chitinase inhibits labeling. The cyst walls of both species contain 3 nm fibrils, which are supposed to be of chitinous nature. In the cyst wall ofB. undulans, several thin layers of WGA-binding fibrils are interspaced with thick layers of other material. InP. dubius, WGA-binding sites are mainly concentrated in the mesocyst, where the microfibrils appear to represent the major component. These results obtained from two phylogenetically distant species confirm that chitin synthesis is an ancestral feature of ciliated protozoa. The amount and distribution of the chitin fibrils may play an important role in the properties and functions of the wall of the resting cyst.     

Phosphorylation is Involved in the Regulation of the Taurine Influx Via the β-system in Ehrlich Ascites Tumor Cells

The role of 3′,5′-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), protein kinase C (PKC) and phosphatases in the regulation of the taurine influx via the β-system in Ehrlich ascites tumor cells has been investigated. The taurine uptake by the β-system in Ehrlich cells is inhibited when PKC is activated by phorbol 12-myristate 13-acetate (PMA) and when protein phosphatases are inhibited by calyculin A (CLA). On the other hand, taurine uptake by the β-system is stimulated by an increased level of cAMP or following addition of N⁶,2′-O-dibutyryl-3′,5′-cyclic adenosine monophosphate (dbcAMP). The effect of dbcAMP is partially blocked by addition of the protein kinase inhibitor H-89, and suppressed in the presence of CLA. It is proposed that the β-system in the Ehrlich cells exists in three states of activity: State I, where a PKC phosphorylation site on the transporter or on a regulator is phosphorylated and transport activity is low. State II, where the PKC phosphorylation site is dephosphorylated and transport activity is normal. State III, representing a state with high transport activity, induced by an elevated cellular cAMP level. Apparently, cAMP preferentially stimulates taurine transport when the β-system is in State II. Received: 8 September/Revised: 9 November 1995     

Cyst wall formation and endogenous carbohydrate utilization during synchronous encystment of Phytophthora palmivora zoospores

Summary Vigorous agitation caused the zoospores of Phytophthora palmivora to undergo rapid synchronous encystment. The rate of encystment was determined by counting the number of cells with an alkali-resistant cyst wall. 50% of the zoospores formed an alkali-resistant cyst wall within 60 sec of agitation; after 120 sec, essentially all zoospores had encysted. The rate of spontaneous encystment in nonagitated suspensions was much slower. The flagella of nearly all zoospores disappeared within 30 sec of agitation, i.e. prior to the formation of an alkali-resistant cyst wall. Zoospores depend on internal reserves for synthesizing their cyst walls. Approximately 70% of the total carbohydrate in motile zoospores was extracted with water after treating the cells with 70% éthnol. During synchronous encystment, this carbohydrate fraction composed largely of glucans decreased markedly while the insoluble carbohydrate fraction (cyst wall glucan) increased correspondingly. Clearly, the conversion of cytoplasmic glucan into wall glucan plays a major role in zoospore encystment.     

Correlation of cellulose synthesis in vivo and in vitro during the encystment of Acanthamoeba

Acanthamoeba castellanii differentiates when placed in a starvation medium. The mature cysts formed are characterized by a cellulosic wall synthesized from endogenous sources during encystment. A particulate enzyme system whose specific activity increases some 30-fold during encystment catalyzes the formation of an alkali-soluble and an alkali-insoluble β-(1 → 4)-glucan (cellulose). The activity in vitro of this enzyme extracted from populations of cells during encystment correlates with the formation in vivo of the mature cyst and the alkali-insoluble β-glucan of the cyst wall. The conclusion is based on the following observations:

1.

1. Both alkali-soluble and alkali-insoluble β-glucans similar to the enzymatic products of the isolated β-glucan synthetase occur in cyst walls.     

Separation and some properties of two acid phosphatases from suspension cultures ofIpomoea sp.

Two acid phosphatases isolated from culturedIpomoea (moring glory) cells were separated by column chromatography on DEAE-cellulose. The two acid phosphatases have different pH optima (pH 4.8–5.0 and 6.0) and do not require the presence of divalent ions. The enzymes possess high activity toward pyrophosphate,p-nitrophenylphosphate, nucleoside di- and triphosphates, and much less activity toward nucleoside monophosphates and sugar esters. The two phosphatases differ from each other in Michaelis constants, in the degree of inhibition by arsenate, fluoride and phosphate and have quantitative differences of substrate specificity. In addition, they also differ in their response to various ions. Issued as NRCC No. 20658     

Localization of phosphatases in young gametophytes ofPolypodium vulgare L.

Summary The distribution of activity of several phosphatases was investigated in filamentous gametophytes of the fernPolypodium vulgare L. High levels of acid phosphatase, alkaline phosphatase, adenosine triphosphatase, and 5-nucleotidase were found in the rhizoids, where they may be concerned in the uptake of substances by the rhizoids. Glucose-6-phosphatase was localized mainly at the base of the rhizoid where it may be involved in the transport of sugars from the protonema to the rhizoid. In the protonema acid and alkaline phosphatases were localized mainly along the transverse walls, particularly along the distal surface, and it is suggested that they may be concerned in movement of substances along the protonema.     

The enzymic composition of the isolated cell wall and plasma membrane of baker''s yeast

A study was made of the enzyme content of the isolated cell walls and of a plasma-membrane preparation obtained by centrifugation after enzymic digestion of the cell walls of baker's yeast. The isolated cell walls showed no hexokinase, alkaline phosphatase, esterase or NADH oxidase activity. It was concluded that these enzymes exist only in the interior of the cell. Further, only a negligible activity of deamidase was detectable in the cell walls. Noticeable amounts of saccharase, phosphatases hydrolysing p-nitrophenyl phosphate, ATP, ADP, thiamin pyrophosphate and PP(i), with optimum activity at pH3-4, and an activity of Mg(2+)-dependent adenosine triphosphatase at neutral pH, were found in the isolated cell walls. During enzymic digestion, the other activities appearing in the cell walls were mostly released into the medium, but the bulk of the Mg(2+)-dependent adenosine triphosphatase remained in the plasma-membrane preparation. Accordingly, it may be assumed that the enzymes released into the medium during digestion are located in the cell wall outside the plasma membrane, whereas the Mg(2+)-dependent adenosine triphosphatase is an enzyme of the plasma membrane. This enzyme differs from the phosphatases with pH optima in the range pH3-4 with regard to location, pH optimum, substrate specificity and different requirement of activators.     

In vitro excystment of the metacercaria of Maritrema arenaria (Digenea: Microphallidae)

Metacercarial cysts of Mantrema arenaria were subjected to a solution containing trypsin and bile salts at 41°C. This treatment induced intense metacercarial activity and after 15 min metacercariae burst through their cyst walls and emerged. Electron microscopy demonstrated that during the process of excystment the inner layer of the cyst wall changed from a compact to a loose fibrous state. Experiments showed that only cysts containing viable metacercariae underwent this change whereas cysts which had been forcibly vacated before treatment did not. This indicated that the structural change of the inner layer of the cyst wall could not be attributed to the excystment medium. Also there was much less acid phosphatase activity in and on the surface of newly excysted metacercariae compared with encapsulated specimens. It was concluded that the excystment medium induced physical activity in, and the release of enzymic material by, the metacercariae. Together these activities rendered the cyst wall soft and susceptible to rupture by physical pressure.     

Histochemical and ultrastructural studies on the metacercarial cyst of Echinostoma revolutum (Trematoda).

Histochemical and ultrastructural studies were made on the metacercarial cyst of Echinostoma revolutum obtained from the kidney of experimentally infected Physa and Lymnaea snails. Ultrastructural studies revealed three cyst walls, an outer, middle and inner. The outer wall was more electron-dense than the middle, and contained coarser granules than those found in the middle layer. The inner wall was lamellated and contained membranous whorls. Collagenous fibers presumably of host origin surrounded the outer cyst wall. The outer and middle cyst walls stained identically with all histochemical procedures used. These walls contained acid mucopolysaccharides and glycoprotein, whereas the inner cyst wall contained glycoprotein. All cyst walls stained positively with a variety of protein stains.     

Mitogenic Activity of the Cell Walls of Mycobacteria,Nocardia, Corynebacteria and Anaerobic Coryneforms

The mitogenic activity of the cell walls prepared from Mycobacterium bovis BCG, Nocardia rubra, Corynebacterium diphtheriae PW8, and four species of Propionibacterium, Corynebacterium parvum ATCC 11829, Propionibacterium acnes C7, Propionibacterium granulosum ATCC 25564 and Propionibacterium avidum ATCC 25577, were investigated. These cell walls were active as mitogens on normal spleen cells, anti-θ sera-treated spleen cells, macrophage-depleted spleen cells of C57BL/6J mice and cortisone-treated thymocytes of C57BL/6J mice. It was also shown that these cell walls were mitogenic on spleen cells and macrophage-depleted spleen cells of congenitally athymic (nude) mice. The above results suggest that the cell walls investigated in this study act as mitogens on both thymus-derived lymphocytes (T-cells) and bone marrow-derived lymphocytes (B-cells).     

The role of phosphatases in the metabolism of Peridinium cinctum,from Lake Kinneret

The annual algal bloom (February–June) in Lake Kinneret consists almost entirely of the dinoflagellatePeridinium cinctum f.westii (Dinophyceae). To clarify the role of phosphatases in the alga, experiments were carried out using cells from culture or from the lake. In culture, as the external ambient orthophosphate (Pi) concentration decreased, alkaline phosphatase activity increased (and to some extent acid phosphatase activity, as well). Hot water extractable P decreased, although molybdate reactive phosphorus (MRP) appeared to be utilized in preference to the non-MRP component of this pool. Alkaline phosphatase inPeridinium collected from the lake as well as cells grown in culture under a high (3–6 mg l^–1) ambient Pi concentration in both continuous light and a 12:12 light-dark cycle, showed a diurnal fluctuation in activity. These results, together with previous observations suggest that the phosphatases inPeridinium are controlled by changes in intracellular phosphorus levels (other than the hot water extractable pool) and/or by other metabolic processes not directly involved in P nutrition.     

Structural and histochemical changes during seed development of Brassica macrocarpa Guss.

During seed formation of Brassica macrocarpa the development of the embryo precedes that of the integuments; structural changes and histochemical changes are associated. Esterases, acid phosphatases, phenols and starch follow a sigmoid pattern, increasing during embryogenesis and decreasing during seed maturation. In the mature seed, esterase activity is localized in the embryo and in the cells of the mucilaginous, aleuronic and hyaline layers. Acid phosphatases are present in the mucilaginous cells, mainly in the column, the cell walls delimiting intercellular spaces of the cortical cylinder and the adhesion areas of the cotyledons. Phenols are scanty in the root apex, mucilaginous cells and the palisade layer, and abundant in the pigmented layer. Starch is absent in ripe seeds which have lipid and protein reserves. The major classes of storage proteins have molecular weights of 21, 22, 27 and 30 KD and accumulate in the late stages prior to complete drying. Esterases and acid phosphatases in mucilaginous cells of the seed integument suggest that these enzymes are involved in hydrolytic processes occurring prior to germination and that mucilages have a metabolic function in seed-soil interactions.     

The Fine Structure of Acanthamoeba astronyxis,with Special Emphasis on Encystment1

ABSTRACT. The fine structure of the trophozoite, encysting cells, and the cyst of Acanthamoeba astronyxis has been examined. In the trophic form a microtubule organizing center was associated with a well developed Golgi complex. During encystment the organelles of the amoeba changed considerably. The profiles of rough endoplasmic reticulum elongated and were often arranged in circles of multilayered concentric systems, enclosing mitochondria, the nucleus, or other inclusions. The mitochondria showed a tendency toward elongation and constriction. One or two nucleolus-like bodies appeared in the nucleus. Lipid droplets increased considerably in amount and were distributed individually or as aggregates. The mature cyst was star-shaped and surrounded by an almost circular exocyst and an endocyst that was closely apposed to the cell membrane. Both walls differed in their thickness and granulation. The exocyst was continuous over the entire cyst, while the endocyst was interrupted by gaps, ostioles. in the region of the rays. Within the ostioles was a bell-shaped structure, the operculum. The latter was composed of a granular material comparable in electron density to that of the endocyst.