Effect of a juvenile hormone analogue on phosphatase activity in pupae of the stable fly, Stomoxys calcitrans.

The alkaline phosphatase activity of stable fly pharate pupae treated with 10 ng of an insect juvenile hormone analogue (JHA), Stauffer R-20458, or untreated pupae was rhythmic and peaked 48 and 96 hr after larval-pupal apolysis. Those treated with 10 μg were not rhythmic and peaked at 48 and 120 hr. Acid phosphatase activity showed a general increase throughout pupal-adult transformation and was three- to four-fold higher than alkaline phosphatase activity. At 24 and 48 hr after larval-pupal apolysis, acid phosphatase was lower in the treated animals, but at 72 and 120 hr, it was higher. Neither inhibition nor enhancement of acid and alkaline phosphatase activity or by three other JHAs could be demonstrated in vitro.     

Experimentally induced elevations in acid phosphatase activity in hemolymph of Biomphalaria glabrata (Mollusca).

The levels of acid phosphatase activity in the hemocytes and serum of the following four groups of the pulmonate gastropod Biomphalaria glabrata were ascertained: (1) those injected with heat-killed Bacillus megaterium, (2) those challenged with sterile distilled water, (3) those shaminjected, and (4) those left untampered. It was determined that challenge with heat-killed bacteria sesulted in significant elevations in acid phosphatase activity in both cells and serum at 1, 2, and 4 hr postinjection, with the level being highest at 2 hr. Injection with water resulted in a significant elevation in cellular enzyme level at 1 hr postinjection but not at 2 and 4 hr; however, there were significant elevations at 2 and 4 hr in serum. This is interpreted to indicate that the elevated intracellular enzyme was subsequently released into serum. Sham injection resulted in significant elevations in acid phosphatase levels in hemocytes at 2 and 4 hr postinjection but no increase of enzyme activity in serum during the course of the experiment. This is interpreted to mean that this hydrolase was not released as a result of sham injection. The source of acid phosphatase was apparently the cytoplasmic granules of granulocytes, which are true lysosomes.     

Quantification of acetylcholinesterase,acid and alkaline phosphatases in the central nervous system of Schizodactylus monstrosus, D. (schizodactylidae:orthoptera). Effect of topical application of the pyrethrum

The newly emerged adult male and female Schizodactylus monstrosus D. were treated with an insecticide, pyrethrum or with petroleum ether in the case of controls. At selected intervals of 6 h, 12 h and 18 h after treatment, the brain and ventral nerve cord and ganglia were homogenized to estimate the activity of acetyl-cholinesterase, and acid and alkaline phosphatase. The resultant activities of these three enzymes showed that: acetylcholinesterase decreased rapidly, and acid and alkaline phosphatase increased significantly after pyrethrum treatment in both brain and ventral nerve cord with ganglia compared with the controls. The activities of alkaline phosphatase showed a marked fluctuation at different post-treatment periods in both the tissues. The results have been discussed in relation to the impact caused by the treated insecticide and its metabolism.     

A cytochemical study of the leaf-gland enzymes of insectivorous plants of the genus Pinguicula

Summary Cytochemical methods have been used to study the distribution of acid phosphatase, esterase, ribonuclease, amylase and protease activity in the stimulated and unstimulated leaf glands of Pinguicula grandiflora, P. vulgaris, P. lusitanica, and P. caudata. Two gland types are present, stalked and sessile. The stalked glands bear a muco-polysaccharide secretion droplet, and are concerned with capture of the prey; the sessile glands are specialised for digestion. In unstimulated glands of both classes, acid phosphatase, esterase and ribonuclease activity is associated with the anticlinal walls of the head cells, which have a characteristic spongy inner surface, comparable with that of transfer cells. Acid phosphatase and esterase activity was also detected in the vacuoles of the head cells of the sessile glands. Substrate film tests showed that amylase is readily released from the stalked glands but not from the sessile ones, while in contrast proteolytic activity is mainly associated with the sessile glands.On stimulation by suitable nitrogenous materials, the glands begin to sectete fluid onto the leaf surface within 1 hr. During the process the enzymes held in the spongy walls are discharged, and activity is also lost from the intracellular sites in the sessile glands.Digestion on the leaf surface and resorption of the products has been followed autoradiographically after feeding of ¹⁴C-labelled protein. Within 2 hr, digestion products enter the leaf, and move towards the margin in the vascular system. Movement out of the leaf begins within 12 hr. Microautoradiographs showed a concentration of products around the bases of the sessile glands and in the cells of the gland head, showing that these glands are involved in resorption as well as secretion.A possible mechanism of gland function is discussed.     

3-phosphoglycerate phosphatase activity in chloroplast preparations as a result of contamination by Acid phosphatase

The presence of a nonspecific acid phosphatase which had high activity with 3-phosphoglycerate as substrate has recently been reported in Spinacia oleracea L. chloroplasts (Mulligan, Tolbert 1980 Plant Physiol 66: 1169-1173). The subcellular localization of this activity has been reinvestigated by differential centrifugation of spinach leaf homogenates. The fraction sedimenting at 1,200g comprised mostly intact chloroplasts and contained more than half the chlorophyll but only 5% of the 3-phosphoglycerate phosphatase activity present in the homogenate. The fraction of the homogenate pelleting at 5,000g contained broken chloroplasts and had considerable 3-phosphoglycerate phosphatase activity. Further purification of the 1,200g pellet fraction on a Percoll step gradient yielded greater than 95% intact chloroplasts, yet the phosphatase activity was reduced more than 15-fold on a chlorophyll basis by this purification.

When the intact chloroplast and cytoplasmic fractions of mesophyll protoplasts were separated by silicone oil filtering centrifugation, the chloroplast fraction contained more than 90% of the chlorophyll but had less than 12% of the 3-phosphoglycerate phosphatase activity. By contrast, more than 60% of the 2-phosphoglycolate phosphatase was recovered in this chloroplast fraction supporting previous evidence that this phosphatase is localized in the chloroplast stroma.

It is concluded that 3-phosphoglycerate phosphatase activity is not localized in the chloroplast but that the activity present in chloroplast preparations results from contamination by acid phosphatase, which either binds to the thylakoid membranes during preparation or is present as some other contaminant in the preparation. Inasmuch as the enzyme acts on a broad range of substrates its presence in chloroplast preparations, particularly when the percentage of intact chloroplasts is low, could produce artifacts in metabolic studies such as measurement of phosphorylation.

     

THE SYNTHESES OF TOTAL MACRONUCLEAR PROTEIN, HISTONE, AND DNA DURING THE CELL CYCLE IN EUPLOTES EURYSTOMUS

Increased alkaline phosphatase activity is induced in certain epithelial cell cultures by hormones with adrenal glucocorticoid activity or their analogues such as prednisolone (Δ^I-hydrocortisone). Enzyme induction occurs in two distinct phases. During the first 12 hr after the addition of prednisolone, there is a small increase in alkaline phosphatase levels. After 15 to 24 hr, the enzyme activity shows a sudden, marked linear rise, reaching a maximum at 60 to 80 hr. Puromycin blocks enzyme induction immediately, even when added during the period of rapid increase of enzyme. Actinomycin D blocks induction when added no later than 8 hr after the addition of prednisolone. On the other hand, Actinomycin D added during the phase of rapid enzyme induction has no effect for at least 12 hr. These findings suggest that de novo protein synthesis is involved in prednisolone induction of alkaline phosphatase and that the RNA messenger for this enzyme is relatively stable.     

Soluble acid phosphatases from the posterior reproductive system of the female housefly, Musca domestica

When the kinetics of the acid phosphatase enzyme system from the posterior reproductive tract of the female housefly, Musca domestica, were studied, enzyme activity was zero order for 2 hr and was temperature-dependent. Orthophosphate release was linearily related to enzyme concentration, and pH optima between pH 3·3 to 3·7, 4·2 to 4·8, and 5·2 to 5·7 were observed. Magnesium and calcium ions were enzyme activators; arsenate, phosphate, fluoride, and hydroxymalonate ions were inhibitors. Sodium azide had little effect. The similarity of activity exhibited on the test substrates indicated that the soluble enzymes corresponded to the non-specific phosphomonoesterases.Disk electrophoresis showed that at least 6 proteins had acid phosphatase activity and were pH-dependent. Also, electrophoresis of extracts of the various structures of the reproductive tract showed that there was a tissue specificity in the distribution of the acid phosphatase isoenzymes.     

A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase

The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 mmN₆,O²′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 mm. Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.     

Leishmania mexicana: a cytochemical and quantitative study of lysosomal enzymes in infected rat bone marrow-derived macrophages

The cellular localization and activity of the lysosomal enzymes acid phosphatase, trimetaphosphatase, and arylsulfatase were studied in rat bone marrow-derived macrophages infected with Leishmania mexicana amazonensis amastigotes. The specific activity of acid phosphatase normalized for protein content was similar in normal macrophages and in isolated amastigotes, whereas the latter were markedly deficient in trimetaphosphatase and arylsulfatase activities. It is thus likely that trimetaphosphatase and arylsulfatase activities detected in infected macrophages were of host cell origin. The activities of the three enzymes, assayed biochemically, varied independently in the infected macrophages. While arylsulfatase activity was unchanged after infection, the activity of acid phosphatase increased by 19, 40, and 94% at 6, 24, and 48 hr, respectively. Trimetaphosphatase activity rose only slightly during the first 24 hr after infection but increased by 74% at 48 hr. The rise in acid phosphatase activity could be accounted for only partially by multiplication of the amastigotes. Thus, as for trimetaphosphatase, these results suggest enhanced macrophage synthesis of acid phosphatase and/or reduced enzyme degradation by the infected macrophages. The reduction in host cell lysosomes previously described (Ryter et al. 1983; Barbieri et al. 1985) was confirmed but appearance of lysosomal enzyme activity in the parasitophorous vacuole is documented in the present report. Thus, Leishmania do not seem to reduce the amount and the activity of host lysosomal enzymes.     

Effect of high dosages of bacterial challenge on acid phosphatase release from Biomphalaria glabrata hemocytes

Specimens of Biomphalaria glabrata challenged with 2 μl of heat-killed Bacillus megaterium at a concentration of 9 × 10⁹ bacteria/ml showed a significant elevation of acid phosphatase activity level at 2 hr post-challenge but there were no significant fluctuations of enzyme activity at any of the other four time intervals post-challenge. Substantiating the earlier hypothesis of Cheng [Amer. Zool., 23, 129–144 (1983b); Mar. Tech. Soc. J., 17, 18–25 (1983c)], it is postulated that if the dosage of the challenging agent is high, most of the recognition sites on the cell, nuclear, and/or lysosomal membranes become saturated and hence are unavailable for the recognition of subsequent challenge. Also, there is a burst of enzyme hypersynthesis and subsequent release which is reflected by a significant elevation of serum enzyme activity level which, in the present case, occurred at 2 hr post-challenge.     

Characterization of Circadian Behavior in the Starlet Sea Anemone,Nematostella vectensis

Background

Although much is known about how circadian systems control daily cycles in the physiology and behavior of Drosophila and several vertebrate models, marine invertebrates have often been overlooked in circadian rhythms research. This study focuses on the starlet sea anemone, Nematostella vectensis, a species that has received increasing attention within the scientific community for its potential as a model research organism. The recently sequenced genome of N. vectensis makes it an especially attractive model for exploring the molecular evolution of circadian behavior. Critical behavioral data needed to correlate gene expression patterns to specific behaviors are currently lacking in N. vectensis.

Methodology/Principal Findings

To detect the presence of behavioral oscillations in N. vectensis, locomotor activity was evaluated using an automated system in an environmentally controlled chamber. Animals exposed to a 24 hr photoperiod (12 hr light: 12 hr dark) exhibited locomotor behavior that was both rhythmic and predominantly nocturnal. The activity peak occurred in the early half of the night with a 2-fold increase in locomotion. Upon transfer to constant lighting conditions (constant light or constant dark), an approximately 24 hr rhythm persisted in most animals, suggesting that the rhythm is controlled by an endogenous circadian mechanism. Fourier analysis revealed the presence of multiple peaks in some animals suggesting additional rhythmic components could be present. In particular, an approximately 12 hr oscillation was often observed. The nocturnal increase in generalized locomotion corresponded to a 24 hr oscillation in animal elongation.

Conclusions/Significance

These data confirm the presence of a light-entrainable circadian clock in Nematostella vectensis. Additional components observed in some individuals indicate that an endogenous clock of approximately 12 hr frequency may also be present. By describing rhythmic locomotor behavior in N. vectensis, we have made important progress in developing the sea anemone as a model organism for circadian rhythm research.     

Okadaic Acid, a Protein Phosphatase Inhibitor, Inhibits Nerve Growth Factor-Directed Neurite Outgrowth in PC 12 Cells

The biochemical mechanisms involved in neurite outgrowth in response to nerve growth factor (NGF) have yet to be completely resolved. Several recent studies have demonstrated that protein kinase activity plays a critical role in neurite outgrowth. However, little information exists about the role of protein phosphatases in the process. In the present study, okadaic acid, a phosphatase inhibitor (specific for types 2A and 1) and tumor promoter, was used to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. PC12 cells cultured in the presence of 50 ng/ml of NGF started to extend neurites after 1 day. After 3 days, 20-25% of the cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC50 of approximately 7 nM. This inhibition was rapidly reversed after washout of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells, indicating that continual phosphatase activity is required to maintain neurites. Taken together, these results reveal the presence of an okadaic acid-sensitive pathway in neurite outgrowth and imply that protein phosphatase plays a positive role in regulating the neuritogenic effects of NGE.     

Temporal Changes of Acid Phosphatase, Arylsulphatase, β-Galactosidase and β-N-ACETYL-d-Glucosaminidase Activities in Subcellular Fractions of Rat Liver

In this paper circadian changes in the liver enzyme activities of rat housed under highly standardized conditions with 12:12 hour light-dark cycle are shown. Activities of acid phosphatase, arylsulphatase, β-galactosidase and β-N-acetyl-d-glucosaminidase in microsomal and lysosomal fractions and crude homogenate were estimated every 4 hr during one 24-hr period. The enzyme activities were related to 1 mg of protein, 1 mg of DNA and 1 g fresh tissue. Daily changes of enzyme activities were found. In case of activity calculated per 1 mg DNA two maxima at 0500 and at 2100 hr were observed, while activity calculated per 1 mg protein showed one maximum at 0500 hr. Activity calculated per 1 g fresh tissue showed the maximum at 0500 hr for each enzyme only in microsomal fraction. As far as acrophase table is concerned for all enzymes and fractions the acrophase occurred during the night. The obtained results are discussed in relation to lysosomal enzymes synthesis process as well as different reference values.     

The induction of lysosomal enzyme activity in the fat body of Calliphora erythrocephala: Changes in the internal environment

The activity of the lysosomal marker enzyme acid phosphatase in the larval fat body of Calliphora erythrocephala increases during development, but not at the same rate throughout the tissue. During the feeding stage, the posterior region has a higher acid phosphatase activity than the anterior region. When the larvae cease feeding on the 5th day of development, the acid phosphatase activity of the inactive anterior lobe increases rapidly in a mosaic-cell pattern. When 4-day-old feeding stage larvae are starved, this increase occurs one day earlier than normally. After the emptying of the gut, the acid phosphatase activity of all the anterior cells both in normal and in starved larvae exceeds that of those in the posterior region.Transplantation experiments indicate that the induction of acid phosphatase activity in the fat body during normal development, especially in the anterior region, is caused by a change in the internal environment when the larvae cease feeding. Both RNA and protein synthesis are involved in this induction process. Inductive factors are present in 5-day-old larvae as well as during formation of the puparium. The competence of the feeding-stage fat cells to develop high acid phosphatase activity is acquired before the actual induction takes place.     

Okadaic acid stimulates the activity of the nerve growth factor-sensitive S6 kinase of PC12 cells.

PC12 pheochromocytoma cells contain at least two different and separable kinases that phosphorylate the S6 protein of the ribosomes. The activity of one of these S6 kinases is increased by treatment of the cells with nerve growth factor and of the other by treatment with epidermal growth factor. Okadaic acid increases the activity of the nerve growth factor-sensitive S6 kinase. The data suggest that the nerve growth factor-sensitive S6 kinase is activated by phosphorylation on serine or threonine residues and is inactivated by either phosphatase 1 or phosphatase 2A, probably the latter.     

Genesis of hadacidin-induced cleft palate in hamster: morphogenesis, electron microscopy, and determination of DNA synthesis, cAMP, and enzyme acid phosphatase.

A morphological, electron microscopic, and biochemical study was undertaken to analyze the genesis of hadacidin-induced cleft palate in hamster fetuses. Gross and light microscopic observations indicated that hadacidin affected the growth of vertical palatal shelves to induce cleft palate. Electron microscopic observations showed that initial hadacidin-induced changes were seen in the mesenchymal cells. Within 12 hr of drug administration, the perinuclear space was swollen and a lysosomal response injury was evident in the mesenchymal cells. Subsequently, 24 hr after hadacidin treatment, lysosomes appeared in the epithelial cells; changes were also seen in the basal lamina which included separation of the lamina densa from the basal cells, duplication of lamina densa, and complete loss of basal lamina. Between 36 and 42 hr post-treatment, the cellular and basal lamina changes subsided, and the epithelium of vertical shelves underwent stratification. Biochemical determination of enzyme acid phosphatase indicated that the levels of enzyme activity in both the control and treated palatal tissues corresponded to the appearance of lysosomes. Measurement of cAMP levels suggested that the peak activity of cAMP corresponded to that of enzyme acid phosphatase and cell injury. The cAMP activity in hadacidin-injured cells, however, was significantly lower in comparison to that of the dying cells of control palates. Hadacidin treatment also affected DNA synthesis in the developing primordia of the palate. It was suggested that hadacidin injures the precursor cells of the palate prior to the appearance of the primordia, and subsequently affects their proliferative behavior, stunting the vertical growth of the palatal shelves and inducing a cleft palate.     

The mode of regulation of the corpus allatum activity during starvation in adult females of the Colorado potato beetle, Leptinotarsa decemlineata (Say)

When two-day-old female Leptinotarsa decemlineata were starved, their corpus allatum activity, as measured by the radiochemical in vitro assay, was significantly reduced after 24 hr. Such a reduction was not observed when the nerve connections between the central nervous system and the retrocerebral complex were severed and the beetles starved up to 5 days. In some experiments, the rate of juvenile hormone biosynthesis in vitro, was substantiated by measurement of the juvenile hormone titre in the haemolymph by physico-chemical methods. It is concluded that intact nervous connections between the central nervous system and the corpora allata are essential for restraining the juvenile hormone biosynthesis during the initial stages of starvation.Corpora allata from 1-day starved insects were considerably stimulated in vitro by farnesenic acid indicating that juvenile hormone synthesis is controlled enzymatically at a stage prior to the final two steps in the pathway. However, on day 5 of starvation, rate-limitation may occur after formation of this intermediate, since farnesenic acid stimulation was much less at this time.Corpora allata of adult females newly emerged from the soil were activated within 4 hr regardless of feeding.     

Histopathology, histochemistry and acetylcholinesterase activity after repetitive administration of fluostigmine to albino rat

Histopathological, histochemical and biochemical investigations were performed on the brain, sciatic nerve, skeletal muscle, heart, liver and kidney of rats which were given 5% of LD50 dose of DFP for 10 days. A decrease in AChE activity, degeneration of neurons and necrotic changes in the nuclei of hypothalamus, degeneration of myelin sheaths in sciatic nerve, a decrease in succinic dehydrogenase activity in the myocardium, and a minimal decrease of acid phosphatase activity (AcPh) in the liver were found. The biochemical determination of AChE level indicated about 30% AChE activity in erythrocytes and tibialis muscle, and 40% in the brain 1 hr after the last dose of the inhibitor and 80% and 50% respectively on the 7th day after poisoning in relation to normal values.     

CELL CYCLE EVENTS IN THE HYDROCORTISONE REGULATION OF ALKALINE PHOSPHATASE IN HELA S3 CELLS

The increase in alkaline phosphatase in asynchronous cultures of HeLa S₃ cells grown in medium supplemented with hydrocortisone is characterized by a lag period of 10–12 hr. Present studies utilizing synchronous cell populations indicate: (a) a minimum of 8–10 hr of incubation with hydrocortisone is necessary for maximum induction of alkaline phosphatase; (b) the increase in enzyme activity produced by hydrocortisone is initiated exclusively in the synthetic phase of the cell cycle; (c) alkaline phosphatase activity does not vary appreciably over a normal control cell cycle. Radioactive hydrocortisone is rapidly distributed into HeLa cells irrespective of their position in the cell cycle, indicating that inductive effects are not governed by selective permeability during the cell cycle. Hydrocortisone-1,2-[³H] diffuses back from the cell into the medium when the cells are incubated in fresh medium containing no hydrocortisone, and the alkaline phosphatase induction, under these conditions, is completely reversible.     

Effect of juvenile hormone on acid phosphatases in the degenerating flight muscles of the Douglas-fir beetle, Dendroctonus pseudotsugae

Changes in acid phosphatase activity were used to estimate flight muscle degeneration in Dendroctonus pseudotsugae. Acid phosphatase positive sites increased numerically as well as in size in flight muscles of beetles attacking the host logs and those maintained on host bark chips. Topical treatment of beetles with a juvenile hormone analogue further increased the phosphatase activity. Juvenile hormone produced similar effects when applied to insects injected with eserine. An increase in the activity of acid phosphatases was also observed after incubation in culture medium with added juvenile hormone. The rôle of nerves and hormones in the degenerating muscles of D. pseudotsugae is compared with that in the permanently degenerating muscles during metamorphosis of some moths.