Alkaline and acid phosphatase in the intestines of rats infected with a virulent strain of Entamoeba histolytica and treated with emetine and Flagyl

The distribution patterns of alkaline phosphatase (EC 3.1.3.1) and acid phosphatase (EC 3.1.3.2) in the intestine of rats inoculated intracaecally with a virulent strain of Entamoeba histolytica and treated with emetine hydrochloride and metronidazole (Flagyl) were studied. The caecum and the large intestine showed a highly significant increase in alkaline phosphatase activity after amoebic inoculation, and the enhanced activity was lowered by emetine and Flagyl treatment. There was no significant increase in acid phosphatase activity either in the caecum and the large intestine or in the small intestine (ileocaecal end). Intracaecal inoculation of bacterial associates alone from E. histolytica cultures did not produce any significant change in the level of these enzymes in the intestine.     

Boophilus microplus: characterization of larval phosphomonoesterases and isolation of subcellular fractions with high phosphatase activity.

Phosphomonoesterase activity was determined for a 115,000g pellet and soluble fractions resulting from a subcellular fractioning of a homogenate of larval Boophilus microplus. Both fractions showed maximum phosphatase activity at pH 5.5 and 10. Acid phosphatase (EC 3.1.3.2) activity was found to be greatest in the soluble fraction. When the reaction rate was plotted against homogenate concentration, the soluble acid phosphatase deviated from the linear relationship. For both fractions different thermostability patterns were obtained, inactlvation beginning for the alkaline phosphatase (EC 3.1.3.1) at 45–55 C. When the effect of substrate concentration on activity was studied, deviations from the typical hyperbolic behavior were observed. Homogenization of larvae with 5 mm EDTA buffer failed to yield a low-speed pellet with high alkaline phosphatase activity, as it is expected if absorptive structures sediment. Moreover, total alkaline phosphatase activity recovered by this method is significantly lower than activity recovered when homogenization is carried out without EDTA. Alternately, homogenization with 10 mM Tris buffer and 0.25 M sucrose gave 27,000g and 115,000g fractions with high phosphatase activity when fractioned by centrifugation. Alkaline treatment of the 115,000g fraction with 10 mM Tris buffer, pH 7.8, failed to separate endoplasmic reticulum contaminants without loss of phosphatase activity. When the 115,000g fraction was centrifuged in a sucrose density gradient, two activity peaks, coincident for both acid and alkaline phosphatases, were obtained. Antigenic analysis showed the existence of similar antigenic determinants in both peaks “immunologically” presented in different ways.     

Kinetic properties, and location of nonspecific phosphomonoesterases in subcellular fractions of fasciola hepatica

Optimal activity was recorded at pH 4.5–5 and pH 9.0–9.5 and specific activity was seen to be 0.013 μmoles of p-nitrophenyl phosphate/min/mg protein at 37 C at pH 4.5 and 0.00169 μmoles at pH 9.0. The ratio of acid to alkaline phosphatase was 7.7:1.0. The K_m for acid phosphatase (EC 3.1.3.2) was 0.5 mM with a V_max of 0.0128 units/mg protein and 0.2mM for alkaline phosphatase (EC 3.1.3.1) with a V_max of 0.00175 units/mg protein. Acid phosphatase activity was optimal at 60 C and alkaline at 37 C. Linearity of enzyme activity was observed with time after the first 15 min of incubation and with homogenate concentration. KCN at 20 mM inhibited 82% of activity at pH 9.0 but also 91.5% activity at pH 4.5. NaF at 10^?2M inhibited 92% of activity at pH 4.5 but had no effect at pH 9.0. The two flukicides rafoxanide and nitroxynil at 20mM had little effect on activity at pH 9.0 and pH 4.5. Enzyme activity at pH 4.5 was found to be greatest in the microsomal fraction with high activity in the lysosomal and soluble fractions. Histochemically, alkaline phosphatase was restricted to the excretory system, vitellaria, and uterus while acid phosphatase was found in the integument and gastrodermis.     

Phosphatase activity in <Emphasis Type="Italic">Amoeba proteus</Emphasis> at pH 9.0

In the free-living ameba Amoeba proteus (strain B), after PAAG disk-electrophoresis of the homogenate supernatant, at using 1-naphthyl phosphate as substrate and pH 9.0, three forms of phosphatase activity were revealed; they were arbitrarily called “fast,” “intermediate,” and “slow” phosphatases. The fast phosphatase has been established to be a fraction of lysosomal acid phosphatase that preserves some low activity at alkaline pH values. The question as to which particular class the intermediate phosphatase belongs to has remained unanswered: it can be either acid phosphatase, or protein tyrosine phosphatase. Based on data of inhibitor analysis, broad substrate specificity, results of experiments with reactivation by Zn ions after inactivation with EDTA, and another localization in the ameba cell than of the fast and intermediate phosphatases, it is concluded that only the slow phosphatase can be classified as alkaline phosphatase (EC 3.1.3.1).     

Phosphatase activity in <Emphasis Type="Italic">Amoeba proteus</Emphasis> at low pH values

In free-living Amoeba proteus (strain B), three forms of tartrate-sensitive phosphatase were revealed by using PAGE of supernatant of the ameba homogenate obtained with 1% Triton X-100 or distilled water and subsequent staining of gels with 2-naphthyl phosphate as substrate (pH 4.0). The form with the highest mobility in the gel turned out to be sensitive to all tested phosphatase activity modulators. Two other forms with the lower mobilities were completely or significantly inactivated not only by sodium L-(+)-tartrate, but also by L-(+)-tartaric acid, sodium orthovanadate, ammonium vanadate, ammonium molybdate, EDTA, EGTA, O-phospho-L-tyrosine, DL-dithiothreitol, H₂O₂, 2-mercaptoethanol, and ions of heavy metals—Fe²⁺, Fe³⁺, and Cu²⁺. Based on results of inhibitory analysis, lysosomal location in ameba cells, and wide substrate specificity of these two forms, it was concluded that they belonged to non-specific acid phosphomonoesterases (AcP, EC 3.1.3.2). This AcP is suggested to have both phosphomonoesterase and phosphotyrosylprotein phosphatase activities. Two ecto-phosphatases were revealed in culture medium, in which amebae were cultivated. One of them was inhibited by the same reagent as the ameba tartrate-sensitive AcP and seemed to be the AcP released into the culture medium in the process of exocytosis of the content of food vacuoles. In the culture medium, apart from this AcP, another phosphatase was revealed; it was not affected by any tested inhibitors of AcP and alkaline phosphatase. It cannot be ruled out that this phosphatase belongs to the ecto-ATPases found in many protists; however, so far its ability to hydrolyze ATP has not yet been proven.     

Fasciola hepatica: the presence of particle-associated and soluble nonspecific acid phosphatases.

The kinetic and physical properties of acid phosphatases in the lysosomal and microsomal fractions of F. hepatica were found to be similar, indicating that they are one and the same enzyme. In contrast, the biochemical properties of the soluble acid phosphatase (EC 3.1.3.2) were quite different from those of the lysosomal and microsomal fractions. This indicated the presence of two distinct forms of the enzyme one particle associated and the other soluble. Electrophoretic heterogeneity of these two types of acid phosphomonoesterase was seen. Two bands of activity were observed in both lysosomal and microsomal fractions and three bands in the soluble fraction.     

A light- and electron-microscopic study of enzymes in the embryonic shell-forming tissue of the freshwater snail, Biomphalaria glabrata

Abstract. The mode of formation of the molluscan exoskeleton is still poorly understood, but studies on adult snails indicate that enzymes involved in vertebrate bone formation also participate in mollusc shell formation. The enzymes peroxidase, alkaline phosphatase, and acid phosphatase are expressed in a constant pattern and help to identify the different zones of the adult shell-forming tissue. The present study evaluates whether the expression of these enzymes is also a tool for the identification of the developing zones of the embryonic shell-forming tissue. Thus, we analyzed the temporal and spatial activity of the above-mentioned enzymes and of tartrate-resistant acid phosphatase in the shell forming tissues in Biomphalaria glabrata. Embryos of different age groups and adults were studied; alkaline phosphatase activity was seen in very young embryos in the shell field invagination prior to the secretion of any shell material, while peroxidase activity was present from the start of the periostracum production. Acid phosphatase, found in considerable amounts in yolk granules and albumen cells, appeared in the embryonic shell-forming tissue in relatively few Golgi stacks. Tartrate-resistant phosphatase was not present in embryos, but was found in adults in the same zone of the mantle edge as acid phosphatase. Using the enzymes as cell markers, the differentiation of the embryonic shell-forming tissue to the different zones of the adult mantle edge could clearly be followed.     

Schistosoma mansoni: Radioautography of colchicine's effect on [3H]proline incorporation into adults in vitro

Adult Schistosoma mansoni were studied radioautographically in order to ascertain the effect of exposures to a fixed concentration of colchicine (5 × 10^?4M) for varying time intervals upon the incorporation of [³H]proline in the tegument. Additionally, a study was made on the effect of varying time exposures of colchicine on the cytochemical localization of alkaline phosphatase (EC 3.1.3.1) in the tegumental invaginations. Worms exposed to colchicine for more than 2 hr preceding addition of the labeled amino acid displayed significant changes in the pattern of distribution. The most profound change was noted in the male tegument where a statistically significant decrease was observed in treated worms. Female worms, on the other hand, failed to display any effect of the drug on the distribution pattern for the times utilized. The distribution of alkaline phosphatase activity was much reduced in the teguments of both sexes. Morphological effects of the drug included disappearance of microtubules from the cytoplasmic connectives, a stacking of RER in the subtegumental cells, and accumulation of discoid granules and membranous bodies in the subtegumental cells. It is hypothesized that the amino acid is associated with the discoid granule at the subtegumental cell level and is ultimately translocated, with the aid of microtubules in the cytoplasmic connectives, to the tegument. Alkaline phosphatase activity is assumed to be associated with the membranous body.     

Ultrastructural observations on the sensory papillae of juvenile and adult Gorgoderina vitelliloba (Trematoda: Gorgoderidae)

Hoole D. and Mitchell J.B. 1981. Ultrastructural observations on the sensory papillae of juvenile and adult Gorgoderina vitelliloba (Trematoda : Gorgoderidae). International Journal for Parasitology11: 411–417. Ultrastructural observations have been made on the juvenile and adult stages of Gorgoderina vitelliloba from Rana temporaria. Four types of sensory papillae occur; button, rosette, ciliated and domed. Button papillae, which contain a ciliary-rootlet but lack a cilium, occur on the oral sucker, dorsal preacetabular surface and the lateral margins of the fluke. Scanning electron microscopical observations reveal that the tegumental protuberances of the papilla are transformed from a spiked or conical appearance in juvenile flukes to a rounded form in adult flukes. Rosette papillae, which also contain a rootlet, occur on the lip of the ventral sucker of both juvenile and adult flukes. Ciliated papillae only occur on the oral sucker of juvenile flukes. Domed papillae, which contain a large area of electron-dense material, occur on the internal surface of the ventral sucker of both juvenile and adult flukes. The functions of papillae and their possible role in the migration of the parasite are discussed.     

Leishmania donovani: action of excreted factor on hydrolytic enzyme activity of macrophages from mice with genetically different resistance to infection

The effect of purified excreted factor from promastigotes of Leishmania donovani upon the activity of four enzymes from lysed peritoneal exudate cells of mice (C3H and C57BL) was determined. There was no demonstrable effect on acid phosphatase (EC3.1.3.2), β-glucuronidase (EC3.2.1.21), and N-acetyl-β-glucosaminidase (EC3.2.1.29), but β-galactosidase (EC3.2.1.23) was inhibited up to 72% after 3 hr of incubation at 37 C. Inhibition of C57BL mouse enzymes was not significantly different from that of C3H mice. Protamine sulfate combined with the highly negatively charged excreted factor of L. donovani to migrate as a single positively charged band on immunoelectrophoresis. Protamine sulfate also reversed the β-galactosidase inhibition, though this was without direct effect on the enzyme. The excreted factor did not change or lose its charge or antigenicity with regard to precipitating antibody, when incubated with extracts of mouse peritoneal exudate cells, splenocytes, or liver homogenate—irregardless of whether the mice had been infected with leishmaniasis for 1 or 2 weeks or were uninfected.     

Schistosomatium douthitti: carbohydrate metabolism in the adults.

Pathways of carbohydrate metabolism in the adults of Schistosomatium douthitti: were investigated. Histochemical reactions for adenosinetriphosphatase (EC 3.6.1.3) glucose 6-phosphate dehydrogenase (EC 1.1.1.49), phosphogluconate dehydrogenase (EC 1.1.1.43), glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), lactate dehydrogenase (EC 1.1.1.27, 1.1.2.3) isocitrate dehydrogenase (EC 1.1.1.41), succinate dehydrogenase (EC 1.3.99.1), malate dehydrogenase (EC 1.1.1.37), cytochrome oxidase (EC 1.9.3.1), and adenosine triphosphatase (EC 3.6.1.3) were found in the adult worms. Glycogen deposits occurred in the parenchyma.Low oxygen tension immobilized the worms. Tartar emetic, sodium cyanide reduced adult motility in vitro. Manometric experiments demonstrated a respiratory quotient of approximately one. Oxygen uptake was completely inhibited by tartar emetic and partially inhibited by sodium fluoracetate and sodium cyanide. Inhibition by sodium fluoroacetate was partially counteracted by citric acid in the medium.Adults demonstrated an oxygen debt following anaerobic incubation. A maximum of 52% of the glucose consumed under aerobic conditions was excreted as lactic acid. Under anaerobic conditions the amount of lactic acid excreted increased. Acids other than lactic acid were also released. Results indicate that although glycolysis is the major pathway, two additional aerobic pathways also exist, one which is cyanide sensitive and the other cyanide insensitive.     

Suppression of glucocorticoid-induced elevation of alkaline phosphatase in C-4-1 cells by inhibitors of 3-hydroxy-3-methylglutaryl-coenzymea reductase and reversal of the suppression by mevalonate

Cell line C-4-a which produces alkaline phosphatase (EC 3.1.1.4) of the placental type in response to glucocorticoids was grown in the presence of inhibitors of mevalonate formation for periods ranging from 1 to 4 days. When C-4-1 cells were incubated in the presence of 25-hydroxycholesterol (1 μM) or compactin (11.6 μM) the induction of alkaline phosphatase by 0.2 μM dexamethasone was supressed. This suppression could be partially prevented by the addition of mevalonolactone to the growing culture. The reversal effect by mevalonate was most evident with compactin, a well known competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. In contrast, the effect of tunicamycin which inhibits N-linked protein glycosylation and also prevents alkaline phosphatase induction by glucocorticoids could not be reversed by mevalonate. These results implicate mevalonate in alkaline phosphatase induction, possibly through its role as a precursor of dolichols.     

Selectable plasmid vectors with alternative and ultrasensitive histochemical marker genes

Three different histochemical marker genes--E. coli beta-galactosidase gene (lacZ), Drosophila alcohol dehydrogenase gene (ADH) and human placenta alkaline phosphatase gene (ALP)--were cloned into a eukaryotic expression vector also containing the neomycin resistance gene. After calcium phosphate transfection and G418 sulfate selection of recipient BALB/c 3T3 cells, stable transfectants were pooled for histochemical staining. The lacZ-bearing cells produce aqua blue staining for beta-galactosidase; ADH-bearing cells, blue-black staining for alcohol dehydrogenase; and ALP-bearing cells, red staining for alkaline phosphatase. Cells carrying different marker genes can be easily differentiated by double-staining protocols. In addition, various photographic films can be used to enhance the colors of specific histochemically tagged cell classes. These plasmid vectors, providing selectability with the neomycin resistance gene and ultrasensitivity of alternative histochemical marker genes, will be very effective in virtually any biological system requiring analyses of multiple cell clones or classes in culture model systems or in situ.     

The effect of host age on Rana temporaria-gorgoderina vitelliloba interactions

Mitchell J. B. 1982. The effect of host age on Rana temporaria-Gorgoderina vitelliloba interactions. International Journal for Parasitology12: 601–604. Two age groups of tadpoles, and newly metamorphosed and adult male Rana temporaria were fed the metacercarial cysts of Gorgoderina vitelliloba. In the younger tadpoles metacercariae died in their cysts. In the older tadpoles excystment took place and juvenile flukes invaded the kidneys, killing the hosts within 72 h. In newly metamorphosed frogs, an immunological response resulted in some of the juvenile flukes in the kidneys being attacked by eosinophils which adhered to and dissolved the tegument, presumably killing the flukes. In contrast, some young frogs were harmed by flukes in their kidneys. Migration away from the kidneys to the bladder took place on about the twelfth day after infection. Juvenile flukes in the kidneys of adult frogs 7 and 14 days after infection, evoked an inflammatory reaction involving polymorphs and lymphocytes. These cells did not appear to damage the parasites.     

Entamoeba histolytica: ultrastructural localization of Ca2+-dependent nucleotidases

Localization of nucleotidases dependent on Ca²⁺ was investigated cytochemically in axenically cultivated trophozoites of Entamoeba histolytica, strain HM-1:IMSS, with an electron microscope. Ca²⁺-dependent ATPase (EC 3.6.1.3) activity was found on the plasma membrane and on the inner surface of the limiting membrane of a few cytoplasmic vacuoles. Ca²⁺-dependent ADPase, Ca²⁺-dependent thiamine pyrophosphatase, and acid phosphatase (EC 3.1.3.2) activities were detected on the inner surface of the limiting membrane of most of the cytoplasmic vacuoles but not on the plasma membrane. Cytoplasmic vacuoles with these enzymatic activities seemed similar in morphological characteristics. Moreover, the reaction product formed by Ca²⁺-dependent ADPase, Ca²⁺-dependent thiamine pyrophosphatase and acid phosphatase was demonstrable on the inner surface of the limiting membrane of vacuoles containing ingested red blood cells. The reaction product formed by these enzymes was also observed on the periphery of ingested red blood cells. The findings suggest that cytoplasmic vacuoles with these enzymatic activities are lysosomal in nature, probably phagolysosomes; therefore, the enzymes appear to be at least partially associated with primary lysosomes of E. histolytica.     

Multiple forms of alkaline phosphatase in untreated and 5-bromo-2'-deoxyuridine-treated choriocarcinoma cells

The alkaline phosphatases present in choriocarcinoma cells, either untreated or treated with 5-bromo-2′-deoxyuridine (BrdUrd), were purified and characterized. Three forms of phosphatase [I, IIa (or IIIa), and IIb (or IIIb)]were isolated from both the untreated and BrdUrd-treated cells. Although BrdUrd induced the synthesis of all three forms of alkaline phosphatase in these cells, the synthesis of forms IIa and IIb was, however, preferentially stimulated. The forms of phosphatase in choriocarcinoma cells resembled each other in their kinetic properties and thermal lability, but differed in their molecular weights and in their electrophoretic mobilities in nondenaturing polyacrylamide gels. All three phosphatases were inactivated by antiserum to term-placental alkaline phosphatase. The alkaline phosphatases from choriocarcinoma cells differed, however, from the enzyme from term placentas in several physicochemical properties. The phosphatases from choriocarcinoma cells had a lower K_m value for p-nitrophenyl phosphate, were more sensitive to inhibition by l-leucine, levamisole, l-p-bromotetramisole, and EDTA, and were more heat-labile. Phosphatase I comigrated with term-placental alkaline phosphatase on nondenaturing polyacrylamide electrophoretic gels, but phosphatases IIa and IIb migrated more slowly. The apparent molecular weights of phosphatase forms I, IIa, and IIb were estimated by gel filtration and polyacrylamide gel electrophoresis to be 115,000, 240,000, and 510,000, respectively. Although three molecular forms of alkaline phosphatase occurred in choriocarcinoma cells, the subunit molecular weight of these phosphatases appeared to be identical to each other and to the subunit of term-placental alkaline phosphatase (63,000 MW). The alkaline phosphatase in choriocarcinoma cells therefore exists in the dimeric, tetrameric, and octameric forms.     

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.     

Enzymatic separation of cis and trans isomers of alicyclic alcohols

It has been discovered that phosphatases [alkaline phosphatase, orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1, and acid phosphatase, orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] display a remarkable geometric specificity in the hydrolysis of cis and trans isomers of monoorthophosphate esters of substituted alicy clicalcohols. While steric hindrances prevent potato acid phosphatase from hydrolysing cis-2-methylcyclohexyl and cis-2-methylcyclopentyl phosphates, the corresponding trans isomers are readily hydrolysed by the enzyme (non-enzymatic, acid-catalysed or base-catalysed hydrolyses of the cis and trans isomers occur at similar rates). Cis isomers of methylcyclohexyl phosphates, in which the methyl group is remote from the hydrolysed ester bond, 3- or 4-, have nearly the same reactivities to phosphatases as their trans counterparts. However, if the methyl group in position 4 is replaced by a bulky substituent, e.g. tert-butyl, phosphatases again hydrolyse only the trans and not the cis isomer. These phenomena afford a simple method for preparative separation of cis and trans isomers of alicyclic alcohols: a mixture of the isomers is first phosphorylated with POCl₃ and then hydrolysed by phosphatase. The trans alcohol formed is extracted with CCl₄, followed by alkaline hydrolysis of the remaining cis-tester and subsequent extraction of the cis alcohol produced.     

Hydrolase histochemistry of the thymus: development and species variations

In the present investigation the localization and activity of alkaline, neutral, and acid hydrolases of the thymus were studied during development of rats and mice and of various adult species using histochemical methods. If different procedures of tissue pretreatment were employed, several inhibition effects and morphological as well as enzyme histochemical artifacts occurred dependent on the mode of tissue pretreatment. After embedding in glycol methacrylate, sections of the thymus showed a better structural preservation than cryostat sections but were accompanied by a drastic decrease of activity and low localization quality of the final reaction products especially in the case of protease studies with 4-methoxy-2-naphthylamine peptides as substrates. Smears of thymic cells facilitated the allocation of enzymes to mobile or fixed cells in the stroma of the thymus. The perivascular localization of aminopeptidase M could only be shown with combined techniques. In comparison, primarily the proteases yielded information on the thymic stroma and in this context especially on the epithelial reticular cells and the stroma proper but also on thymocytes (lymphocytes) and enabled a species-dependent subdivision of the thymic reticulum already in the light microscope. Enzyme histochemically the development of the rat and mouse thymus could be subdivided into an early period and perinatal (pre- and postnatal) period of functional differentiation. Morphological (proliferation of cortical lymphocytes) and enzyme histochemical changes (disappearance of dipeptidylpeptidase IV, significant loss of alkaline phosphatase activity and beginning activity increase of aminopeptidase M) occurred primarily at the transition from the early to the prenatal period. During the postnatal phase, a significant activation of lysosomal enzymes in the thymic medulla and general enzymatic differentiation of the cortical epithelial reticular cells were found. Species differences and species similarities for the respective enzymes and their localization as well as for the thymic cells were noticed for adult rats, mice, guinea-pigs, hamsters, and marmoset monkeys. Differences were true especially for the thymocytes; less species differences were seen for the epithelial reticular cells; capsular and perivascular connective tissue and the macrophages behaved rather similarly. Species-independently certain medullary epithelial reticular cells showed high and typically localized alkaline phosphatase activities and species-dependently also high activities of neutral hydrolases.(ABSTRACT TRUNCATED AT 400 WORDS)