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.     

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.     

The ultrastructure of cell wall formation and of gamma particles during encystment of Allomyces macrogynus zoospores

Structural changes during cell wall formation by populations of semisynchronously germinating zoospores were studied in the water mold Allomyces macrogynus. Fluorescence microscopy using Calcofluor white ST (which binds to -1,4-linked glycans) demonstrated that Calcofluor-specific material was deposited around most cells between 2–10 min after the induction of encystment (beginning when a wall-less zoospore retracts its flagellum and rounds up). During the first 15 min of encystment there was a progressive increase in fluorescence intensity. Ultrastructural analysis of encysting cells showed that within 2–10 min after the induction of encystment small vesicles 35–70 nm diameter were present near the spore surface, and some were in the process of fusing with the plasma membrane. The fusion of vesicles with the zoospore membrane was concomitant with the appearance of electron-opaque fibrillar material outside the plasma membrane. Vesicles similar to those near the spore surface were found within the gamma () particles of encysting cells. These particles had a crystalline inclusion within the electron-opaque matrix. During the period of initial cyst cell wall formation numerous vesicles appeared to arise at the crystal-matrix interface. Approximately 15–20 min was required for the cell wall to be formed. We suggest that the initial response of the zoospore to induction of encystment is the formation of a cell wall mediated by the fusion of cytoplasmic vesicles with the plasma membrane.Non-Standard Abbreviations GlcNac N-Acetylglucosamine - DS sterile dilute salts solution - PYG peptone-yeast extract-glucose broth     

Microcyst formation in the plasmodial slime moldDidymium iridis

Summary Myxamoebae ofDidymium iridis were removed from the bacterial food source and induced to encyst by transfer to 10 mM phosphate buffer. After 24 hours of induction approximately 90% of the myxamoebae had differentiated into microcysts. The kinetics of encystment were not significantly affected by pH or osmolarity of the encystment medium. Early stages of encystment were distinguished by the appearance of autophagic vacuoles and an extracellular slime-like sheath. The outer wall layer, consisting of dense fibrils, was unevenly deposited after 4 hours. An electron-lucent, second wall layer appeared between 5–10 hours followed by a densely packed, third wall layer adjacent to the plasma membrane. Wall formation appeared to involve smooth-membraned vesicles of possible Golgi origin. The vesicle contents and outer wall layer reacted with the periodic acid-silver methenamine stain for polysaccharide. The density of intramembrane particles of the protoplasmic fracture face increased during encystment with a gradual formation of aggregates of particles.Florida Agricultural Experiment Station Journal Series No. 3473.     

Decreased ouabain binding in cystic fibrosis fibroblasts in potassium-free medium

We have previously demonstrated that cystic fibrosis (CF) cells show increased survival compared to normal cells when exposed to ouabain in medium lacking potassium. In this report, we show that the CF cells bind significantly less ouabain than the normal cells in potassium-deficient medium. Using age-matched normal and CF skin fibroblast strains, we show that (1) at ouabain concentrations <20 × 10⁻⁹ M binding for both normal and CF cells is linear with time for 1 h and reaches equilibrium after 4 h; (2) at ouabain concentrations between 2 and 20 × 10^-9 M, the initial rate of binding for CF cells is approx. 70% of the normal cells; (3) under equilibrium-binding conditions, Scatchard analysis reveals that three different CF strains have 12, 16, and 44% fewer ouabain-binding sites than their matched normal controls. In addition, studies in potassium-free medium of the inhibition of ⁸⁶Rb flux (a K⁺ analogue) into the cell by ouabain show no differences between CF and normal cells. We have also previously shown that in a low glucose, potassium-deficient medium the CF cells survived ouabain exposure no better than normal cells. In this report, equilibrium-binding studies with [³H]ouabain clearly show that CF cells bind less ouabain under these conditions than normal cells. These results indicate that ouabain resistance in CF cells is not solely a function of differences in ouabain binding. Furthermore, the differential ouabain killing may not be due to ion transport differences, but rather to as yet unknown mechanisms. CF cells thus appear to be unlike previously characterized ouabain-resistant mutants.     

Morphological and biochemical differentiation of limb bud cells cultured in chemically defined medium

When chick limb buds were isolated from stage 22–23 embryos and cultured in chemically defined medium “BGJb,” the explants grew and, after about 9 days, showed good chondrogenesis of recognizable cartilage segments. Cartilage-type proteoglycan (termed PCS-H) was not synthesized during early days of culture, but by Day 9, it became a major proteoglycan constituent of the tissue. Freshly dissociated limb bud cells, when plated as monodispersed cultures at a density of 7 × 10⁶ cells/ml of BGJb, did not undergo chondrogenic differentiation and, instead, assumed the appearance of unhealthy or degenerated cells. During 9 days of culture, even though proteoglycans were synthesized, they were nevertheless of much smaller molecular size than PCS-H. When limb bud cells were cultured as a pellet containing 7 × 10⁶ cells in 1 ml of BGJb, a more tightly packed aggregate of about 2 × 10⁶ cells appeared in an inner region of the pullet during the first 24 hr of culture, and by Day 12 the aggregate had differentiated into a cartilage nodule surrounded by a thin layer of what appear to be ectodermal cells. As the conversion of aggregate into cartilage nodule progressed, newly formed proteoglycans gradually became more like cartilage-type proteoglycans, and by Day 12 they had many chemical and physical characteristics similar to those of the proteoglycans isolated from fully differentiated cartilages. The results indicate that the initial association of limb bud cells is an important factor for the chondrogenesis in BGJb and further suggest that the tight binding of the cell surfaces to one another may directly or indirectly stimulate the mechanism of synthesis of cartilage-type proteoglycans.     

Bacterial conjugation betweenEscherichia coli andPseudomonas spp. donor and recipient cells in soil

Summary Experiments conducted in microcosms containing loam soil samples inoculated with eitherE. coli orPseudomonas spp. donor and recipient cells showed that bacterial cells survived and conjugated over a 24-h incubation period.E. coli transconjugants were detected 6 h after donor and recipient strains were introduced into sterile soil samples. In non-sterile soil samples, transconjugants were detected between 8 and 24 h incubation.Pseudomonas transconjugants were recovered from sterile soil samples between 6 and 12 h after their introduction and as early as 2 h in non-sterile soil. The results show that genetic interactions occur in non-sterile soil in relatively short periods of time at relatively high transfer frequencies (10^–3 to 10^–4). Studies on genetic interactions in soil are becoming necessary in risk assessment/environmental impact studies prior to the release of genetically engineered or modified organisms into uncontained environments.     

Giardia intestinalis: Expression of ubiquitin, glucosamine-6-phosphate and cyst wall protein genes during the encystment process

We determined the relative expression of ubiquitin (ub), glucosamine-6-phosphate-isomerase (gn6pi) and cyst wall protein (cwp) genes during encystment of the Portland-1 and Portland-1R strains of Giardia intestinalis. Encystment was induced with bile for different time periods. The presence of encystment-specific vesicles (ESVs) and the relative expression of genes (log₁₀ΔRn) were determined by transmission electron microscopy and real-time PCR, respectively. Our results demonstrated the gene expression and the presence of ESVs after 6 h of encystment. Values of cwp2 gene expression increased by 591-fold in strain Portland-1 and 78.2-fold in strain Portland-1R at this time point compared to values at 0 h, after which values gradually decreased until reaching basal values between 8 and 18 h after the encystment started. Expression of gn6pi was 43.5- and 46.3-fold higher than basal values, in Portland-1 and Portland-1R, respectively. Ub gene expression was 82.25-fold higher than its basal levels at 4 h, after which expression decreased gradually until reaching basal values after 16 h. Conclusions: This work showed the relationship between the presence of ESVs and encystment gene expression at 6 h, and resistance to albendazole does not inhibit the encystment process. The results revealed important knowledge with implications in the control of parasite dissemination for preventing parasite transmission.     

Synchronization of encystment of <Emphasis Type="BoldItalic">Scrippsiella lachrymosa</Emphasis> (Dinophyta)

The encystment of Scrippsiella lachrymosa cells (strain B-10), which can be induced reliably in encystment medium, was inhibited by stirring the culture. 100 mL cultures in glass beakers were stirred at 1 rotation s⁻¹. Stirring inhibited vegetative cells from congregating (swarming) at the walls of the culture container. When stirring was stopped, a rapid induction of sexual reproduction was seen. As soon as stirring stopped (within 2 min), cells were observed swarming near the edges of the glass beaker. Four days after cessation of stirring, large percentages of the cells were mating and, after 7 days, most were zygotes. Cultures were observed after 31, 38, and, 45 days of stirring. When cultures were stirred for 45 days, cysts developed in the stirred treatments, but these cysts were attached to flocculent material that had also formed in the medium. The use of this laboratory method is advantageous for the study of the mating through cyst stages of the dinoflagellate life history. This method may also demonstrate the need for a ‘surface’ as a place for the dinoflagellate to congregate in order to successfully encyst and may help explain environmental observations of encystment at pycnoclines.     

Effect of dibutyryl cyclic AMP on the restoration of contact inhibition in tumor cells and its relationship to cell density and the cell cycle

KB cell cultures exposed to 10⁻⁴ M dibutyryl cyclic AMP were significantly inhibited and exhibited contact inhibition of growth at cell densities of 8 × 10⁴/cm² irrespective of the initial plating density. Control cultures reached densities of 2.5 × 10⁵/cm². Inhibition of growth did not occur in KB cells when the density was below 1 × 10⁴ cells/cm². When dibutyryl cyclic AMP was removed from KB cells in the contact-inhibited state, growth resumed with DNA synthesis beginning in about 6 h. Labeled metaphases increased rapidly after 22 h without the appearance of an early rise in unlabeled metaphases. This suggests that the inhibitory effect of dibutyryl cyclic AMP is on the G1 phase of the cell cycle.     

A comparative study of in situ hybridization procedures using cRNA applied to Drosophila hydei salivary gland chromosomes

The effect of different denaturation and hybridization procedures on the efficiency of in situ ³H-cRNA hybridization with DNA in the polytene chromosomes of Drosophila hydei was investigated.Denaturation of the DNA in the squash preparations with 90% formamide in 0.1 × SSC at 65 °C for 2.5 h gave a significantly higher retention of radioactivity following in situ hybridization than did denaturation by 30 sec incubation in boiling 0.1 × SSC.A comparison of the effect of various SSC concentrations in the hybridization mixture revealed that among the SSC concentrations tested, 3 × SSC or 4 × SSC gave the highest efficiency of hybrid formation.Hybridization in 50% formamide at 20 °C resulted in continuing hybrid formation over a period of 3.5 h, the majority of the cRNA/DNA hybrids being formed within the first 10 min of the incubation period. The thermal dissociation profile of in situ cRNA/DNA hybrids formed in 50% formamide, 4 × SSC at 20 °C, as determined in 0.1 × SSC indicated a T_m of 66 °C. The shape of the profile and the results of competition experiments suggested a high fidelity of base-matching in the in situ ³H-cRNA/DNA hybrids.Non-chromosomal background labeling in autoradiographs of polytene chromosomes hybridized with ³H-cRNA was effectively reduced by adding a 200–1000 fold excess of cold 28S + 18S RNA.     

Axenic cultivation of Naegleria gruberi : Requirement for methionine

A simplified axenic medium for Naegleria gruberi strain NEG-M contains -methionine, dextrose, yeast extract, a macromolecular fraction of fetal calf serum, and phosphate buffer. Amoebae cultured in suspension in this medium grow with doubling times of 8–10 h (at 32 °C) to yield 2–4 × 10⁶ cells/ml. Amoebae from growing or early stationary phase cultures, transferred to nonnutrient buffer, differentiate synchronously into flagellates. Differentiation occurs reproducibly 80 min after initiation (time for 50% flagellates at 25 °C) if amoebae are taken from a culture maintained at pH 6.6.     

Poly(U) binding to ascites cells of the 13762A rat mammary adenocarcinoma: Evidence for a protein receptor on the cell surface

Ascites cells of the 13762 rat mammary adenocarcinoma bind poly(U) in a reaction that is complete within 5 min at 0°C. Poly(U) binding is saturable; the capacity of these cells is 5×10⁷ UMP residues/cell (approx. 2×10⁵ chains/cell). Most [³H]poly(U) bound in the rapid reaction can be recovered in an undergraded state. However, it is rapidly degraded by low concentrations of exogenous pancreatic ribonuclease. The magnitude of binding is independent of temperature and ionic conditions, and is unaffected by metabolic inhibitors or concanavalin A (ConA). Radioactivity presented as [³H]poly(U) tends to co-fractionate with 5′-nucleotidase after homogenization of cells in the media of low ionic strength, but is efficiently released from cells exposed to protein denaturants that effectively fix cellular RNA in situ. Cells pretreated with proteolytic enzymes have sharply reduced capacities to bind poly(U). Autoradiography of cells bearing [³H]poly(U) demonstrates a uniform distribution of radioactivity through the cell population and is consistent with binding to the plasma membrane. These and other results imply that binding of poly(U) to 13762 ascites cells is mediated by protein receptors on the cell surface.     

Synchronisation de cellules BHK 21 par carence partielle en -asparagine

The isolation of mutants requiring -asparagine from a population of BHK 21/13 cells has enabled us to synchronize the cell growth using the deprivation effect of this amino acid. In the absence of asparagine, cells cannot enter a new cycle of DNA synthesis and accumulate in G 1 phase, If asparagine is added to 24 h growth-arrested cultures, a wave of DNA synthesis is observed after a 6 h latency period, followed by a partially synchronous cell division reaching a 55% labelling index. During a 24 h period, in the presence of very small amounts of asparagine (1 to 6 × 10⁻⁶ M) a higher cell fraction accumulates at G 1 period. In these conditions the degree of synchronisation is improved, reaching an 85% labelling index, following a 24 h incubation in the presence of 6 × 10⁻⁶ M asparagine.L'isolement de mutants exigeants en -asparagine d'une population clonale de cellules BHK 21/13 nous a permis d'exploiter l'effet de la carence en cet acide aminé pour synchroniser les cellules. En l'absence d'asparagine, les cellules ne peuvent entrer dans un nouveau cycle de synthèse de DNA et s'accumulent en phase G 1. Après 24 h de carence, si l'on place les cellules ainsi quiescentes dans un milieu contenant de l'asparagine, on observe après un temps de latence de 6 h une onde de synthèse de DNA suivie d'une synchronisation partielle des divisions cellulaires. En présence de faibles quantités d'asparagine (1 à 6 × 10⁻⁶ M) un plus grand nombre de cellules s'accumulent en phase G 1 durant la période de carence de 24 h. Dans ces conditions l'enrichissement de cette phase en cellules nous a permis d'améliorer le degré de synchronisation et d'obtenir un index de marquage de 85% après une carence en 6 × 10⁻⁶ M d'asparagine.     

Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium

A chimaeric gene has been constructed that expresses -D-glucuronidase (GUS) in transformed plant tissues, but not in bacterial cells. This gene has proved extremely useful for monitoring transformation during the period immediately following gene transfer from Agrobacterium tumefaciens. GUS expression was detectable 2 days after inoculation, peaked at 3–4 days and then declined; if selection was imposed expression increased again after 10–14 days. The extent of transient expression after 4 days correlated well with stable integration as measured by kanamycin resistance, hormone independence, and gall formation. Histochemical staining of inoculated leaf discs confirmed the transient peak of GUS expression 3–4 days after inoculation. The most surprising result was that the blue staining was concentrated in localized zones on the circumference of the disc; within these zones, essentially all the cells appeared to be expressing GUS. We suggest that the frequency of gene transfer from Agrobacterium is extremely high within localized regions of leaf explants, but that the frequency of stable integration is several orders of magnitude lower.     

Immunohistochemical analysis of inflammatory infiltrates around the injection site of a streptococcal preparation,OK-432, in rat tongue

On the assumption that neutrophils around the injection site of OK-432, a heat- and penicillin-treated lyophilized preparation of the Su strain ofStreptococcus pyogenes, enhance immunologic response through the production of Interleukin-1 (IL-1), OK-432 was injected into rat tongue, and specimens from the tongue were immunohistochemically investigated at various intervals after the injection, to clarify the process of inflammatory and immune responses at the injection site. Neutrophils and mononuclear cells appeared around the OK-432 injection site after 1 hour, increased to their maximum level at 24 hours, and then decreased from the 3rd to the 7th day. IL-1 was detected on neutrophils 3 hours after the injection, and OX-08-positive cells (suppressor/cytotoxic T cells and the majority of natural killer cells) remarkably increased. OX-39-positive cells (IL-2 receptor) appeared after 12 hours. These results suggest that neutrophils around the injection site of OK-432 at early phases of inflammation play a role in the expression of BRM function through IL-1.     

Rearrangement of concanavalin A receptor sites on cells tagged with dinitrofluorobenzene: Evidence for the chemical induction of a change usually associated with malignant transformation

Normal human peripheral blood granulocytes which are tagged with 1-fluoro-2,4-dinitrobenzene (DNFB) are agglutinated by concanavalin A (ConA) in a way which resembles the pattern of reactivity displayed by leukemic cells. The present study further defines this reaction. The binding of ConA to untagged and DNP-tagged granulocytes, treated with DNFB at a ratio of 10¹¹ molecules/cell, was quantified by isotopic dilution experiments employing [³H]ConA. Similar amounts of the lectin were bound to untagged and DNP-tagged cells following incubation for 5 min at 4 °C or 30 min at 24 °C: 1.1 × 10⁵ molecules/cell, 4.6 × 10²/μ² of surface area, and 1.6 × 10³/μg of protein. The binding of [³H]ConA to both untagged and DNP-tagged cells was inhibited to the same degree by α-methylglucopyranoside (α-MG). Fixation with either glutaraldehyde or formaldehyde, which immobilizes ConA receptor sites, completely inhibited the agglutination of both untagged and DNP-tagged cells although lectin binding was unchanged. This suggests that the inhibition of agglutination was not due to the blocking of ConA-binding sites by aldehyde groups but rather to the immobilization of lectin receptors. We conclude that dinitrophenylation of normal granulocytes facilitates the rearrangement of lectin receptors in a way which resembles the ConA-induced clustering of sites which have been observed with malignant and transformed cells.     

Adenylate cyclase activity during growth and encystment of Acanthamoeba castellanii

The adenylate cyclase activity of Acanthamoeba castellanii (Neff) was studied in extracts prepared after breaking cells in the Potter-Elvehjem homogenizer. The adenylate cyclase activity of cells is low during the exponential growth phase, but then rises 2–4-fold during the stationary phase to a peak, roughly at the time that cyst forms are detectable in the culture. A 2–4-fold activity rise to a peak also occurs 4–8 h after late log cells are transferred to a non-nutrient encystment medium, a time which is shortly before numbers of cyst forms can be detected in the culture. The pattern of activity observed when stationary phase cells are transferred to encystment medium is complex and depends in part on whether the cultures have exhibited the peak of cyclase activity and have begun to initiate cyst formation prior to the transfer. Within the usual time frame after transfer to encystment medium, early logarithmic phase cells do not exhibit a 2–4-fold rise of cyclase activity and they do not encyst. The results suggest a relationship between encystment and the pattern of rise and fall in cyclase specific activity. Fractionation of the homogenate of trophozoites indicated that adenylate cyclase activity was associated with the particulate microsomal fraction.     

Staphylococcal Footpad Infection in Mice

Local footpad infection in mouse was investigated with 55 clinically isolated strains of Staphylococcus aureus. When 10⁷ viable cells were inoculated into the footpad, local swelling and bacterial growth resulted after 24 hr. With a dose of 10⁶ cells, moderate swelling was observed after a few hours but the reaction had almost disappeared after 24 hr. About 75% of the staphylococcal strains tested caused footpad edema in mice at doses of 10⁷ cells. A statistical comparison of the virulence of the organisms on intravenous and intraperitoneal injection with that in inducing footpad swelling is also reported.     

MULTIPLE ROUTES OF SEXUALITY IN ALEXANDRIUM TAYLORI (DINOPHYCEAE) IN CULTURE1

Alexandrium taylori Balech is a cyst‐forming dinoflagellate species responsible for recurrent blooms in Mediterranean coastal waters. The nuclear development of the cells during the sexual cycle and the effect of different external nitrate and phosphate levels were studied. Nuclear fusion of gametes occurred 6–12 h after the complete cytoplasmic fusion. The U‐shaped nuclei fused through the end of one nucleus and the mid‐area of the other. The mobile and biflagellated zygote had a large, U‐shaped nucleus and may follow three different fates: direct division, short‐term encystment (ecdysal), and long‐term encystment (resting). Ecdysal cysts may divide in >24–96 h into two, four, six, or eight cells before germinating. Meiosis presumably occurred in three locations: in the planozygote, within the ecdysal cyst, and in the planomeiocyte (germling) liberated either from ecdysal or resting cysts. The effects of nutrients on these routes were studied in individually isolated sexual stages. (1) Direct divisions occurred mainly under replete conditions (L1), whereas no direct planozygote divisions were recorded in media with no phosphate added (L‐P). (2) Short‐term encystment was larger in media lacking phosphate (L‐P and L/30) than in medium with no nitrate added (L‐N) or under replete conditions (L1). (3) Long‐term encystment was only observed in medium with no nitrate added (L‐N). The long‐lived resting cyst, not previously described for this species, had a clear double wall, an irregular shape, a flat morphology, and a middle orange spot. No cysts germinated in 1–2 months, whereas 86% of the cysts germinated 2–3 months after being formed. A flow cytometry analysis showed that sexual induction and zygote formation were very fast and highly common processes, zygotes being nearly half of the population at days 3 and 5 after the induction of sexuality in the cultures.