Morphological and Developmental Studies of the Snake Trypanosome Trypanosoma hydrae Ayala, Atkinson, Vakalis, 1983 in Experimentally Infected Hosts and in Culture

Trypanosoma hydrae from the broad-banded watersnake, Nerodia fasciata confluens, underwent development in the freshwater leech, Placobdella parasitica. Epimastigotes and transitional stages were present only in the crop and gastric caecum. Only one metacyclic form was observed. The potential of leeches as vectors is discussed. Two broad-banded watersnakes were infected by inoculation with culture forms of T. hydrae maintained on NNN medium. Parasitemias varied from 10⁵/ml to 10⁶/ml with dividing forms seen only rarely. A broad and a slender form of T. hydrae are described from the peripheral blood of a watersnake 7 months after an experimental infection.     

Relationship between sperm count, serum gonadotropins and testosterone levels in normo-, oligo- and azoospermia

In 92 men with normozoospermia (greater than 40 X 10(6)/ml), 105 with slight oligozoospermia (greater than 10 X 10(6)/ml), 100 with severe oligozoospermia (less than 10 X 10(6)/ml) and 56 with azoospermia, serum testosterone, LH and FSH were measured radioimmunologically. With an increasing degree of reduction of spermatozoa, a decreasing testosterone level and increasing LH and FSH levels could be demonstrated. In normozoospermia, between 40 and 140 X 10(6)/ml, a direct correlation was found between FSH and sperm count, and, in the group between 40 and 100 X 10(6)/ml, a direct correlation between T and sperm count. A disturbed LH:T balance is often observed which beside decreased serum T levels demonstrates a testicular deficiency in androgen production.     

DNA adduct formation, metabolism, and morphological transforming activity of aceanthrylene in C3H10T1/2CL8 cells

Aceanthrylene (ACE), a cyclopenta-fused polycyclic aromatic hydrocarbon (CP-PAH) related to anthracene, has been studied for its ability to be metabolized, to form DNA adducts, and to morphologically transform C3H10T1/2CL8 mouse embryo fibroblasts in culture. Although ACE has been previously shown to be a strong mutagen in Salmonella typhimurium strains TA89 and TA100, it did not transform C3H10T1/2 cells (0.4-16 micrograms/ml) under 2 treatment protocols: treatment (for 24 h) 1 day after seeding the cells; treatment (for 24 h) 5 days after seeding the cells. Both protocols are effective in detecting the morphological transforming activity of PAH and CP-PAH and the latter protocol has been shown to be effective in detecting chemicals which are active in the first protocol only with the additional treatment of the cells with a tumor promoter. ACE is metabolized by C3H10T1/2 cells to ACE-1,2-dihydrodiol (the cyclopenta-ring dihydrodiol) at a rate of 450 pmoles ACE-1,2-dihydrodiol formed/h/10(6) cells. ACE-7,8-dihydrodiol and ACE-9,10-dihydrodiol, identified as major Aroclor-1254-induced rat liver microsomal metabolites from their UV, NMR, and mass spectral data, were not identified in incubations of C3H10T1/2 cells with ACE. ACE-DNA adducts in C3H10T1/2 cells were isolated, separated, identified, and quantitated using the 32P-postlabeling method. ACE forms 4 major adducts and each was identified as an ACE-1,2-oxide/2'-deoxyguanosine adduct. The level of adduction was 2.18 pmoles ACE adducts/mg DNA after a 24-h incubation of ACE (16 micrograms/ml) with C3H10T1/2 cells. ACE-DNA adduct persistence and repair were evaluated in C3H10T1/2 cells using a hydroxyurea block after ACE treatment. ACE-DNA adducts were not repaired under the conditions used in the morphological transformation studies. Thus, ACE provides an interesting example of a mutagenic PAH which is metabolized by C3H10T1/2 cells to active intermediates, forms relatively stable and persistent 2'-deoxyguanosine adducts in C3H10T1/2 cells, and yet induces no detectable morphological transforming activity under the experimental conditions used.     

Enterobacteriaceae, coliforms, faecal coliforms and salmonellas in raw ewes' milk

Thirty-nine samples of freshly drawn ewes' milk collected at 13 farms, and 120 samples of raw ewes' milk collected on arrival at a dairy were examined. Farm samples had geometric mean counts of 4.4 X 10(2) Enterobacteriaceae/ml, 3.9 X 10(2) coliforms/ml and 2.0 X 10(2) faecal coliforms/ml, whereas the respective mean counts were 6.2 X 10(3)/ml, 5.4 X 10(3)/ml and 1.3 X 10(3)/ml for dairy samples. Salmonellas were not detected by enrichment procedures in any of the 159 samples examined. Escherichia coli (47.5% strains), Enterobacter cloacae (17.7%), Ent. agglomerans (11.3%), Hafnia alvei (6.5%) and Klebsiella oxytoca (6.0%) were the predominant species in 434 Enterobacteriaceae strains isolated from farm samples. Levels and species of Enterobacteriaceae found in the present work in raw ewes' milk imply a considerable risk of early blowing in cheese-making from unpasteurized milk.     

Pathogenicity of Spiroplasma taiwanense for larval Aedes aegypti mosquitoes

Helical replicative forms, but not the persistent non-replicative forms, of Spiroplasma taiwanense Abalain-Colloc et al. (isolated from the mosquito Anopheles sinensis Wiedemann in Taiwan) were shown to reduce significantly the survival of Aedes aegypti (L.) mosquito larvae reared in 10 ml of water with 0.3 ml of S.taiwanense suspensions added on days 0 and 3. The suspensions contained, respectively, helical forms at a concentration of 10(9) Colour Change Units (CCU)/ml and persistent forms at 10(6) CCU/ml. It is suggested that S.taiwanense, or toxins produced from it, are potentially useful for use in integrated mosquito control programmes.     

Growth response, reproductive activity, and serum growth hormone in fine-wool ewe lambs treated with triiodothyronine

Forty-four 6-mo-old, fine-wool ewe lambs were randomly allotted to one of three triiodothyronine (T(3)) treatment groups (14 or 15 ewe lambs/group). Treatments (0, 200, and 400 mug T(3)) were administered daily by subcutaneous injection during a 30-d treatment period in September. Following the treatments, ewe lambs were allowed to mate with Debouillet rams during a 34-d breeding season. Animal weights did not differ (P > 0.10) among treatment groups throughout the study. However, ewe lambs receiving 400 mug T(3)/d gained less (P < 0.05) during the treatment period than did those receiving 200 mug T(3)/d or controls. Grease fleece weights (6-mo clip) were similar (P > 0.10) among treatment groups. Serum growth hormone (GH) values in blood samples collected on Days 7 and 28 of the 30-d treatment were similar (P > 0.10) before treatments were administered and on Day 7 after treatments were administered. However, on Day 28 after treatments were administered, serum GH was lower (P < 0.01) in ewe lambs receiving 400 mug T(3)/d (1.5 ng/ml) than in those receiving 200 mug/d (2.1 ng/ml) or controls (2.2 ng/ml). Ovarian cyclicity was determined by monitoring serum progesterone. Forty percent of control ewe lambs were determined to have cycled during treatment compared with 20% of those receiving 200 mug T(3)/day and none of those receiving 400 mug T(3)/d (P < 0.10). Pregnancy rates were lower (P < 0.10) in ewe lambs receiving 400 mug T(3)/d (29%) compared with those receiving 200 mug T(3)/d (60%) or controls (66.7%). Day of conception, lambs born/ewe exposed, lambs born/ewe lambing, and lambs weaned/ewe lambing did not differ (P > 0.10) among treatments. Preweaning performance of offspring was not altered by maternal treatment with 200 or 400 mug T(3)/d (P > 0.10). These data indicate that 200 and 400 mug T(3)/d will not enhance GH production or improve reproductive performance of ewe lambs.     

Induction of microtubule-damage, mitotic arrest, Bcl-2 phosphorylation, Bak activation, and mitochondria-dependent caspase cascade is involved in human Jurkat T-cell apoptosis by aruncin B from Aruncus dioicus var. kamtschaticus

Exposure of human Jurkat T cells to aruncin B, purified from Aruncus dioicus, caused apoptosis along with microtubule damage, G(2)/M-arrest, Bcl-2 phosphorylation, Bak activation, mitochondrial membrane potential (Δψm) loss, cytochrome c release, activation of multiple caspases, and PARP degradation. Analyses by employing Bcl-2 overexpression and selective caspase inhibitors revealed that G(2)/M-arrest and Bcl-2 phosphorylation occurred prior to mitochondria-dependent activation of caspase-9, -3, and -8. The IC(50) values for human resting T cells, activated T cells, and Jurkat T cells were >60μg/ml, 49μg/ml, and 22μg/ml, respectively. These results demonstrate the apoptogenic activity of a novel microtubule-damaging agent aruncin B.     

High concentration active enzyme centrifugation studies with pig kidney phosphofructokinase. Detection of 9.8 S, 25 S, and 53 S active polymeric forms

This laboratory has carried out the first detailed studies of the active polymeric forms of phosphofructokinases over the concentration region of 1 to 1200 micrograms/ml. This includes the concentration range in which the enzymes exist in vivo and the concentration range in which their association-dissociation equilibria shift to yield various polymeric forms. Previously, active enzyme centrifugation experiments were limited to the concentration range below a few micrograms per ml. The present experiments were made possible by the recent development in this laboratory of a new technique called high concentration active enzyme centrifugation (Wei, G. J., and Deal, W. C., Jr. (1979) Biochemistry 18, 1129). We report here three new active polymeric forms of pig kidney phosphofructokinase which have been observed in high concentration active enzyme centrifugation experiments. These include: 1) a 9.8 S form (Mr = 2.6 X 10(5)); 2) a 25 S form (Mr = 1.01 X 10(6)); and 3) a 53 S form (too asymmetric to estimate Mr). In addition, a 5.4 S form is predicted from the Mr (8.8 X 10(4)) of the polypeptide chain obtained from sodium dodecyl sulfate gel electrophoresis; it is not known whether or not it is active. The 9.8 S value is the limiting sedimentation coefficient value observed in active enzyme centrifugation experiments. The 25 S form is indicated by a plateau in the 50 to 200 micrograms/ml region of the s versus c curve. The 53 S form is observed as a plateau in the 600 to 1000 micrograms/ml region of the s versus c curve.     

Effects of sperm concentrations and culture media on fertilization and development of in vitro matured pig oocytes

This study was carried out to investigate the effects of sperm concentrations and culture media on fertilization and development of in vitro matured pig oocytes. The concentrations of frozen-thawed sperm were 0.2 x 10(7), 2 x 10(7), 20 x 10(7) and 200 x 10(7)/ml, respectively. Culture media were NCSU-23, HEPES-buffered (25 mM) NCSU-23, PZM-3 and PZM-4, respectively. Increasing the sperm concentration from 0.2 x 10(7) to 2 x 10(7)/ml, significantly increased the penetration rate. Also, increasing the sperm concentration from 20 x 10(7) to 200 x 10(7)/ml increased the penetration rate from 62.1% to 69.9%, respectively, with no differences between these two concentrations. A similar pattern was observed for polyspermic penetration and male pronucleus formation. The mean number of sperm per oocyte significantly increased in the 20 x 10(7)/ml and again in the 200 x 10(7)/ml sperm concentrations. The percentage of blastocysts from cleaved oocytes at the 2 x 10(7)/ml sperm concentration was significantly higher than that at the 0.2 x 10(7), 20 x 10(7) and 200 x 10(7)/ml sperm concentrations. The percentage of blastocysts from cleaved oocytes and the cell numbers per blastocyst were significantly higher in the HEPES-buffered NCSU-23 culture medium than in the NCSU-23, PZM-3 and PZM-4 culture media under a gas atmosphere of 5% CO2 in air.     

Trypanosoma acomys (Wenyon, 1909): continuous culturing with a mouse fibroblast cell-line (A9)

The continuous culturing of Trypanosoma acomys in the presence of a murine areolar-adipose cell line (A9) was possible for the 1st time. The trypanosomes were cultured at 37 degrees C with A9 in DMEM supplemented with 20% heat inactivated fetal bovine serum, using an initial inoculum from primary cultures of lung or blood clots from infected spiny mice. The cultures were maintained for 115 days and underwent 15 passages before termination and cryopreservation. Using this culture system T. acomys subcultures were initiated from 3 different initial inocula (3 x 10(4), 1.5 x 10(5) and 7.4 x 10(5) parasites/ml) and growth curves revealed that the lowest inoculum gave the best growth pattern. This inoculum yielded a population doubling time of less than 12 h for 4 days, a high peak density of 7 x 10(6) parasites/ml and the most gradual decline compared to the other 2 inocula. Rosetting epimastigotes and nests of amastigotes were observed in close association with the feeder layer cells. Epimastigotes were the most predominant form in culture supernatants but other morphological forms observed included trypomastigotes and sphaeromastigotes.     

Inhibition of growth hormone-stimulated lipolysis by somatostatin, insulin, and insulin-like growth factors (somatomedins) in vitro

The effects of somatostatin, insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II)/MSA on growth hormone (GH) (1 microgram/ml)-induced lipolysis were examined employing chicken adipose tissue in vitro. Basal and GH-stimulated glycerol release were inhibited by somatostatin (1 ng/ml) and by IGF-II/MSA (10 and 100 ng/ml). Insulin and IGF-I (10 and 100 ng/ml) completely inhibited the lipolytic response to GH without affecting basal glycerol release. Insulin and IGF-I were equipotent in inhibiting GH-induced lipolysis while IGF-II is only 16% as potent as insulin.     

The use of metronidazole, tinidazole and dimetridazole in eliminating trichomonads from laboratory mice

Metronidazole, tinidazole and dimetridazole were administered in the drinking water for 5 days to mice experimentally infected with Tritrichomonas muris and Tetratrichomonas microta. Mice were successfully infected with T. muris and T. microta recovered from infected gerbils. The trichomonas infection was successfully eliminated in mice given a 1% sucrose solution containing 2.5 mg/ml metronidazole or tinidazole. Mice receiving 1.0 mg/ml metronidazole, 1.0 mg/ml tinidazole and 1.2, 5.0 and 10.0 mg/ml dimetridazole failed to eliminate the trichomonas organism. A reduction in water intake was only noted with mice receiving 10 mg/ml dimetridazole. In mice receiving only 1% sucrose the infection was not eliminated.     

Changes in plasma testosterone, thyroxine and triiodothyronine in relation to sperm production and remex moult in domestic ganders

Changes in plasma testosterone (T), thyroxine (T4), triiodothyronine (T3), semen output and remex moult were studied in domestic ganders. A bimodal pattern in both plasma T and sperm concentration was observed during the annual cycle. Ganders started to produce semen at the end of January; maximum semen volume (0.32 +/- 0.04 ml) and sperm concentration (148 +/- 38 x 10(3)/mm3) were reached in March and a marked decrease was observed after mid-April, when the moult of the remiges began. Plasma T3 levels peaked in February (9.7 +/- 0.6 nmol.l-1) and this peak coincided with maximum T concentrations (9.8-10.4 nmol.l-1). Elevated levels of T4 were found from late February until mid-April (31.0-33.6 nmol.l-1). Plasma T concentration was low at all stages of remex moult and regrowth. Decreased T4 levels were found in ganders during remex regrowth from the "brush" to half of the full primary growth stage. Higher plasma T4 levels were found before and after this stage of the moult. A reverse pattern was observed for T3 concentrations.     

Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays

A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrP(C) to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD(50)). Brain tissue from 263K scrapie-affected hamsters gave SD(50) values of 10(11)-10(12)/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD(50) values of 10(3.5)-10(5.7)/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD(50) values of 10(2.0)-10(2.9)/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.     

Binding of lectins to culture and vector forms of Trypanosoma rangeli Tejera, 1920 (Protozoa, Kinetoplastida) and to structures of the vector gut

Culture forms of Trypanosoma rangeli could be agglutinated with Canavalia ensiformis (Con A) lectin and, less effectively with Pisum sativum agglutinin (PEA), at a concentration of 200 micrograms/ml. Ricinus communis agglutinin I (RCA I) agglutinated trypanosomes only if they were not previously washed with physiological Ringer's solution. Three other lectins did not react with the same parasite forms. Direct or indirect lectin-gold labeling techniques were applied to LR-White embedded thin sections of T. rangeli culture forms and to forms in the gut, hemolymph, and salivary glands of Rhodnius prolixus. Under these conditions, Con A was the only lectin out of 9 that bound to the surface of trypanosomes from culture and from the bug hemolymph. Con A did not react with any midgut or salivary gland forms. The preservation of the biological activity of the lectin-gold complexes that did not bind to the parasite surface was confirmed by reactions with structures of the invertebrate host.     

Effect of bovine herpesvirus-1 or bovine viral diarrhea virus on development of in vitro-produced bovine embryos.

In previous experiments, zona pellucida (ZP)-intact in vitro-produced (IVP) embryos incubated for 1 hr with 10(6.3) TCID(50)/ml bovine herpes virus-1 (BHV-1), 10(5.3) TCID(50)/ml cytopathic (CP) bovine viral diarrhea virus (BVDV) or 10(5.3) TCID(50)/ml noncytopathic (NCP) BVDV showed no signs of virus replication or embryonic degeneration. The aims of the present study were to investigate whether a prolonged presence (24 hr or 8 days) of 10(6.3) TCID(50)/ml BHV-1 or 10(5.3) TCID(50)/ml BVDV in an in vitro embryo production system affected the rate of cleavage and embryonic development of ZP-intact embryos, and to point out eventual causes of adverse effects. When virus was present in each step of an IVP system, significantly lower rates of cleavage and blastocyst formation of virus-exposed embryos were observed, in comparison with control embryos (P < 0.01). When embryos were only exposed to virus during the in vitro fertilization (IVF), the rates of cleavage and blastocyst formation were significantly affected. The introduction of BHV-1 or BVDV during in vitro maturation (IVM) or in vitro culture (IVC) resulted only in significantly lower rates of blastocyst (P < 0.01). In all experiments, virus replication was not detected in the embryonic cells. On the other hand, virus replication was clearly demonstrated in oviductal cells in the co-culture system, resulting in a degeneration of these cells. In an additional experiment, synthetic oviduct fluid (SOF) without somatic cells was used as an alternative culture system. Even when SOF-embryos were exposed to 10(6.3) TCID(50)/ml BHV-1 or 10(5.3) TCID(50)/ml CP, and NCP BVDV, the rates of blastocyst formation of the BHV-1-, CP-, and NCP BVDV-exposed embryos were not different from the unexposed control embryos, 23%, 24%, and 24%, respectively, vs. 27%. Taken together, it can be concluded that the virus-induced adverse effects on embryonic development in conventional co-cultures were due to changes in the embryonic environment caused by infection of oviductal cells.     

甲肝病毒在人血白细胞中体外连续传代和在分类白细胞中增殖的研究

1985年我们采用间接免疫荧光法(IF法)检测出甲肝患者外周血白细胞中有甲肝抗原(HAAg)存在,继而又将HAAg阳性白细胞直接种入PLC/PRF/5细胞,分离到两株甲肝病毒(HAV)NJ—3株和H—1株。为了弄清白细胞所携带的病毒究竟仅为吸附吞饮,抑或能在其中复制增殖,我们将分离到的HAV用正常人血白细胞进行体外增殖试验,现将结果报告如下。     

Binding of Lectins to Culture and Vector Forms of Trypanosoma rangeli Tejera, 1920 (Protozoa, Kinetoplastida) and to Structures of the Vector Gut

Culture forms of Trypanosoma rangeli could be agglutinated with Canavalia ensiformis (Con A) lectin and, less effectively with Pisum sativum agglutinin (PEA), at a concentration of 200 μg/ml. Ricinus communis agglutinin I (RCA I) agglutinated trypanosomes only if they were not previously washed with physiological Ringer's solution. Three other lectins did not react with the same parasite forms. Direct or indirect lectin-gold labeling techniques were applied to LR-White embedded thin sections of T. rangeli culture forms and to forms in the gut, hemolymph, and salivary glands of Rhodnius prolixus. Under these conditions, Con A was the only lectin out of 9 that bound to the surface of trypanosomes from culture and from the bug hemolymph. Con A did not react with any midgut or salivary gland forms. The preservation of the biological activity of the lectin-gold complexes that did not bind to the parasite surface was confirmed by reactions with structures of the invertebrate host.     

Isolation and regeneration of protoplasts from the yeast and mycelial form of the dimorphic zygomycete Benjaminiella poitrasii: role of chitin metabolism for morphogenesis during regeneration

Experimental parameters for isolation and regeneration of protoplasts from the mycelial and yeast form cells of the dimorphic zygomycete Benjamininiella poitrasii are reported. Using a chitosanase containing preparation from Streptomyces sp. MCl we obtained protoplasts after 5 h incubation with a yield of 2+/-0.3 x 10(6) ml(-1) and 3+/-0.4 x 10(7) ml(-1) for the mycelial and yeast form, respectively. During regeneration under conditions triggering dimorphism the two morphological forms were observed after 36 h. Initially, for 10-12 h only an irregular mass was formed as a result of deregulated cell wall synthesis. Among the tested inhibitors influencing cell wall metabolism, chitin metabolism inhibitors showed distinctive effects on the regeneration of protoplasts suggesting that the respective enzymes significantly contribute to determining the morphogenesis of the dimorphic fungus B. poitrasii.     

Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers

Blood stream forms (BSF) of Trypanosoma brucei brucei GUT at 3.1 were propagated in vitro in the absence of feeder layer cells at 37 C, using a modified Iscove's medium (HMI-18). The medium was supplemented with 0.05 mM bathocuproine sulfonate, 1.5 mM L-cysteine, 1 mM hypoxanthine, 0.2 mM 2-mercaptoethanol, 1 mM sodium pyruvate. 0.16 mM thymidine, and 20% (v/v) Serum Plus (SP) (Hazleton Biologics, Lenexa, Kansas). The latter contained a low level of serum proteins (13 micrograms/ml). Each primary culture was initiated by placing 3.5-4 x 10(6) BSFs isolated from infected mice in a flask containing 5 ml of the medium (HMI-9) supplemented with 10% fetal bovine serum (FBS) and 10% SP. The cultures were maintained by replacing the medium every 24 hr for 5-7 days. During this period, many BSFs died. However, from day 4 onward, long slender BSFs increased in number. On days 5-7, trypanosome suspensions were pooled and cell debris was removed by means of diethylaminoethyl cellulose (DE52) column chromatography. Blood stream forms then were collected by centrifugation, resuspended in fresh medium at 7-9 x 10(5)/ml, and transferred to new flasks. Subcultures were maintained by readjusting the BSF density to 7-9 x 10(5)/ml every 24 hr. Concentrations of FBS were reduced gradually at 5-7-day intervals by alternating the amounts of FBS and SP in HMI-9 with 5% FBS and 15% SP, with 2% FBS and 18% SP, and finally with 20% SP (HMI-18). By this method, 2-3 x 10(6) VSFs/ml were obtained consistently every 24 hr. for more than 80 days.(ABSTRACT TRUNCATED AT 250 WORDS)