Destruction of Enteric Bacteria in Liquid Egg with β-Propiolactone

Liquid whole egg or egg white, inoculated with Escherichia coli 1485, Salmonella senftenberg ATCC 8400, or Salmonella typhimurium 84-I, was treated with concentrations of β-propiolactone ranging from 0.05 to 0.3%. Egg white containing 1 × 10³ to 1 × 10⁶ cells of E. coli 1485 per ml was sterilized in 1 hr at 27 C by lactone concentrations of 0.2 and 0.3%. Egg white containing 1 × 10⁵ cells of S. senftenberg ATCC 8400 per ml was sterilized in 12 hr at 10 C by 0.1% lactone and in 2 to 3 hr by 0.3% lactone at the same temperature.

Liquid whole egg inoculated with 1 × 10⁵ cells of either species of Salmonella was sterilized in 4 to 5 hr at 10 C with 0.2% lactone or in 2 to 3 hr by 0.3% lactone at this temperature. A mild heat treatment of either 15 min at 37 C or 1 min at 55 C markedly shortened the exposure times required for sterilization by β-propiolactone at 10 C.

After disinfection was complete, the lactone-treated liquid whole egg was reinoculated with low cell numbers of either species of Salmonella to determine the presence of residual lactone or toxic products. Liquid whole egg treated with 0.2% lactone would support the growth of salmonellae after 13 to 14 hr at 10 C. A heat treatment of 45 min at 37 C or 10 min at 55 C immediately after addition of 0.2% lactone allowed growth of the salmonellae in the lactone-treated liquid whole egg. No evidence of residual toxicity from the lactone treatment was found.

The amount of lactone needed to prevent the outgrowth of low cell numbers of either strain of Salmonella in liquid whole egg was quantitated. Liquid whole egg containing 0.06 to 0.07% lactone would not support salmonellae growth from inocula of 1 to 10 cells per ml of egg. Lactone concentrations above 0.08% prevented outgrowth of salmonellae inocula of 10 to 200 cells per ml of liquid whole egg.

     

Schistosoma mansoni: degradation of host extracellular matrix by eggs and miracidia

A radioactively labeled in vitro model of the extracellular matrix of the mammalian intestinal wall and of snail tissue was used to determine whether proteolytic enzymes released by eggs and miracidia of Schistosoma mansoni could degrade connective tissue macromolecules in the type of interactive framework found in vivo. Eggs were collected and miracidia hatched in the presence of antibiotics to eliminate bacterial contamination. Uninfected livers were used as controls to ensure that the tissue dissociation and egg collection procedures did not produce proteolytic activity. One thousand live eggs incubated with the extracellular matrix for 72 hr at 37 C degraded 31% of the glycoprotein in the matrix; there was no degradation of elastin or collagen. Medium conditioned by incubation with eggs degraded 60% as much of the matrix as the live eggs themselves. The proteolytic activity of the egg-conditioned medium was greater in the presence of dithiothreitol. Miracidia incubated with the extracellular matrix in tissue culture medium at 27 or 37 C rapidly transformed to living sporocysts. This transformation was accompanied by a release of proteolytic activity, resulting in the degradation of 49 to 58% of the glycoprotein in the extracellular matrix by 1000 miracidia. Again, no elastin or collagen was degraded. The time course of degradation by miracidia was rapid over 24 hr and thus similar to that previously reported for cercariae. Degradation by eggs occurred more slowly over 72 hr. These data confirm that both eggs and miracidia secrete proteinases which are capable of degrading at least the glycoprotein components of extracellular matrix to facilitate their migration through intestinal wall or penetration of snail tissue.     

Use of Antibiotics in the Preparation of Canine Kidney Tissue Culture Vaccines to Eliminate Leptospiral Infection Hazards

The potential leptospiral infection hazard in the use of vaccines prepared from canine kidney monolayer cultures was studied. Cell cultures were prepared from kidneys of dogs experimentally infected with Leptospira serotype canicola. Viable leptospires were found in kidney cell suspensions at the time of seeding, surviving trypsinization either at room temperature for approximately 2 hr or overnight at 4 C, even in the presence of antibiotics. In tissue cultures maintained without antibiotics, leptospires were cultured up to the time of involution of cells at 25 to 34 days of incubation. Cytopathogenic effects of leptospires on cultured kidney cells were not noted; neither was growth of leptospires remarkable. Generally, the leptospire culture titer decreased to 10^-4 or 10^-5 at the 4th hr or 1st day of incubation to 10^-1 or negative by the 30th or 34th day of incubation. The addition of either a combination of penicillin (100 units per ml) plus streptomycin (100 μg/ml) or polymyxin B (50 units per ml) plus dihydrostreptomycin (100 μg/ml) to seeding cell suspensions resulted in the elimination of viable leptospires by the 4th hr of incubation. From cell cultures treated with neomycin (100 μg/ml) or chloramphenicol (100 μg/ml), leptospires were recovered, respectively, after 24 and 48 hr, but not thereafter. It was apparent that antibiotics, particularly the combination of polymyxin B and dihydrostreptomycin, could be effectively used to eliminate leptospires in tissue culture. Other antibiotics with known antileptospiral activities probably would be effective also. If antibiotics are not used in canine kidney tissue culture employed for viral vaccine preparations, rigid testing for the presence of leptospires in donor dogs and tissue-culture vaccine is indicated.     

Chromosome Preparations in the Field from Mammals Long After Death

Chromosome preparations of high quality can be obtained from bone marrow cells of small mammals that have been dead for 20 hr or longer. The bone marrow is rinsed out of the femurs with RPMI medium supplemented with 15% fetal calf serum. Add 0.05-0.1 ml of a 0.01 % colchicine solution to 5 ml of medium-cell suspension. After Vi-1 hr of colchicine treatment at 37 C the cells are spun down and the supernatant replaced by 5 ml of hypotonic (0.075 M) KC1. After 12 min in the hypotonic solution at 37 C the cells are fixed in methanokacetic acid 3:1. Air dried preparations are made after repeating the fixation procedure three times and the chromosomes are stained with Gietnsa, if required after prclieatment of the preparations for banding; e.g., GTG. Technical hints for field work are given. The technique has proven successful with several species of rodents and shrews.     

Chromosome preparations in the field from mammals long after death

Chromosome preparations of high quality can be obtained from bone marrow cells of small mammals that have been dead for 20 hr or longer. The bone marrow is rinsed out of the femurs with RPMI medium supplemented with 15% fetal calf serum. Add 0.05-0.1 ml of a 0.01% colchicine solution to 5 ml of medium-cell suspension. After 1/2-1 hr of colchicine treatment at 37 C the cells are spun down and the supernatant replaced by 5 ml of hypotonic (0.075 M) KCl. After 12 min in the hypotonic solution at 37 C the cells are fixed in methanol:acetic acid 3:1. Air dried preparations are made after repeating the fixation procedure three times and the chromosomes are stained with Giemsa, if required after pretreatment of the preparations for banding, e.g., GTG. Technical hints for field work are given. The technique has proven successful with several species of rodents and shrews.     

Halophilic Bacteria Susceptibility to Peracetic Acid Vapor and Ethylene Oxide

Extremely to slightly halophilic bacteria were tested for susceptibility to two sterilizing agents, peracetic acid (PAA) and ethylene oxide (ETO). PAA susceptibility was explored by two methods: an agar plate (constant pH of 7.2) and a filter strip (constant incubation period of 37 days); 100% susceptibility was obtained by both methods. The dosage (0.5 ml/min) was applied to a filter pad in a petri dish cover. Glove box experiments with ETO (input 1.5 lb. [ca. 680.4 g]/24 hr, the only constant) yielded 100% susceptibility for all halophiles tested. These experiments demonstrated the efficacy of two lethal agents for extreme halophiles, PAA and ETO. Variation in pH did not affect susceptibility.     

In vitro explant culture of mantle epithelium of freshwater pearl mussel

The in vitro culture of nacre secreting pallial mantle explants of freshwater pearl producing mussel, Lamellidens marginalis (Lamarck) included depuration of pearl mussels with different physical and chemical agents to eradicate various commensals, removal of pallial mantle ribbon, aseptic preparation of explants from the ribbon and transfer of those explants into tissue culture petri dishes. Special synthetic tissue culture media enriched with additives viz., inactivated calf fetal serum and antibiotics were poured into plates with explants. The culture plates were incubated at 30 degrees C in a CO2 incubator at 5%, CO2. The cultures could be maintained for 42-45 days without any contamination. After 12 hr epithelial like cells began to migrate out and formed a complete cell sheet surrounding the explant within 12-15 days. The epithelial cells in the culture indicated functional viability as subsequently after 38-40 days of culture, typical aragonitic 'nacre' crystals of CaCO3 could be observed throughout the culture plates.     

Nano silver treatment is effective in reducing bacterial contaminations of Araucaria excelsa R. Br. var. glauca explants

The downside of plant tissue culture techniques is an unwanted microbial contamination. Elimination of contaminants is the first step of any successful investigation on plant tissue culture. Preliminary experiments on Araucaria excelsa R. Br. var. glauca (Norfolk-Island pine) (syn.: A. heterophylla) showed that most common decontaminants could not successfully eliminate the contamination. Therefore, nano silver (NS) colloids were evaluated for controlling contamination. Treatments were included soaking the explants in NS solution or adding NS to the culture medium. Explants were cultured on MS medium supplemented with appropriate growth regulators for their establishment. Results showed that surface sterilization followed by treatment with 200 mg l-1 of NS with soaking time of 180 min reduced the bacterial contamination from 61.5% to 11.3% and adding 400 mg l-1 NS to the medium reduced the bacterial contamination from 81.25% to 18.75%. Nano silver could be applied without adverse effects on plant growth and development. This is the first report on in vitro establishment of A. excelsa R. Br. using NS to reduce bacterial infections.     

Increase in activity of glucose 6-phosphate dehydrogenase in mouse mammary tissue cultured with insulin

1. In organ cultures of mammary tissue from C3H mice we observed increases in the activity of glucose 6-phosphate dehydrogenase similar to that occurring at parturition. 2. In 22hr. cultures of tissue from late-pregnant mice insulin was required for the increases, but the further addition of prolactin, corticosterone and certain other hormones had no effect. The rise in activity occurred over the second half of the culture period. 3. Results from culture of adipose tissue, and mammary tissue rich in adipose tissue, strongly suggest that the rise in activity occurs in mammary parenchymal rather than adipose cells. 4. In 45hr. cultures prolactin prevented a fall in enzyme activity between 22hr. and 45hr. If the medium contained serum the activity at 22hr. was unaffected, but it continued to rise up to 45hr., and prolactin then had no effect. 5. The enzyme also increased in activity in cultures of mammary tissue from mid-pregnant mice. Insulin was again required, the activity was higher at 45hr. than at 24hr. and prolactin increased the activities at both these times. 6. Actinomycin D, cycloheximide and puromycin at low concentration in the media of 22hr. cultures all prevented increases in enzyme activity. Hydroxyurea at a concentration that inhibited the incorporation of [(3)H]thymidine into DNA by 92% had little effect. 7. Actinomycin D and cycloheximide largely failed to prevent the rise in enzyme activity if added after 3.5hr. and 12hr. respectively. Hence all essential RNA and protein synthesis appears to be finished by 3.5hr. and 12hr., although most of the increase in enzyme activity occurs gradually between 12hr. and 22hr. 8. We suggest that the increases in enzyme activity, both in culture and in the living animal at parturition, are induced by an influx of glucose that is restrained during pregnancy by the growth-hormone-like action of placental lactogen.     

No evidence for effects of mild microwave irradiation on electrophysiological and morphological properties of cultured embryonic rat dorsal root ganglion cells

Effects of mild microwave treatment (1 hr, 37 degrees C) on the in vitro development of rat mechanically dissociated dorsal root ganglion (DRG) neurons were investigated to establish whether microwave irradiation effects exist on nervous tissue other than heat induced tissue fixation. Phase contrast microscopy and immunocytochemical neurofilament stainings did not reveal significant differences between irradiated (2 hr after isolation) and control cultures, maintained up till 21 days. The electrophysiological properties of microwave exposed and non-exposed DRG neurons were compared using the whole-cell patch-clamp technique. Control neurons, in culture for 0-12 days, were excitable. In cultured cells (1-12 days), microwaved 2 hr after isolation, the action potentials were similar to or slightly different from those of the control cells. No acute microwave effects were found on neurons irradiated after 1 day of culture. These results suggest that mild microwave irradiation has neither significant acute nor strong long-term effects on DRG culture development and DRG neuron membrane properties, consistent with the notion that microwave effects essentially are temperature effects.     

Immunofluorescent Detection of Enterotoxin B in Food and a Culture Medium

Both Staphylococcus aureus strains 243 and S-6 cells producing enterotoxin B and free enterotoxin in food and culture medium were rapidly demonstrated by using the fluorescent-antibody technique. Comparison of cell fluorescence and enterotoxin B production determined by double gel diffusion showed that an estimation of enterotoxin production could be made by observing the degree of cell fluorescence. The fluorescent-antibody technique was used to determine whether cells were producing enterotoxin under varying nutritional and environmental conditions: NaCl concentration, culture aeration, and time and temperature of incubation in Brain Heart Infusion broth and shrimp slurries. At the various NaCl concentrations, the fluorescence of cells was found positively associated with enterotoxin B production only during the first 12 hr of growth. As the NaCl concentration was increased from 0 to 10%, the fluorescence of cells and toxin production decreased. Maximum for cell fluorescence and enterotoxin production was observed at 37 C. Little or no difference in cell fluorescence and enterotoxin production with both strains was found between Brain Heart Infusion broth and shrimp slurry cultures. All results obtained with the fluorescent-antibody technique were verified with double gel diffusion for enterotoxin detection and quantitation.     

Determination of Amino Acid Sequence in Surfactin,a Crystalline Peptidelipid Surfactant Produced by Bacillus subtilis

Some conditions of autolysis in cultured tobacco cells were examined for temperature, cell culture age and aeration. Cells autolyzed readily at 45°C. Seventy percent of the dry matter, almost 100% of the soluble sugar, 40% of the insoluble sugar and 60% of the total nitrogen in the initial cells were excreted within 5 hr of incubation in water. At lower physiological temperatures, excreted substances were reabsorbed into cells during the early period of incubation under aerobic conditions.

Rapidly growing cells excreted larger amounts of sugar, nitrogen and solid matter than did non-growing cells during autolysis at 30°C.

Plasmolysis was observed in autolyzed cells.

Autolysis was makedly stimulated by anaerobic conditions.     

Trypanosoma cruzi: in vitro interactions between cultured amastigotes and human skin-muscle cells.

Established cultures of human skin-muscle cells were used for determining the parasite—host cell relationship of Trypanosoma cruzi amastigotes (12–16 passages) cultured in a cell-free medium (F-69) at 37 C. The medium used for this experiment was tissue culture fluid M-199 enriched with 10% fetal bovine serum and relatively high concentrations of ATP, ADP and AMP. Amastigotes entered skin-muscle cells incubated at 32 or 35 C, multiplied and completed their intracellular life cycle in about 7 days. At 35 C, 23.6% of cells became infected in 7 days and at 32 C, 43.6% were infected in 5 days. The higher infection rate of cultured cells at 32 C was probably due to more frequent and prolonged cell-parasite contact, as amastigotes multiplied in the tissue culture medium and remained viable for a longer period at the lower temperature. As a control, epimastigotes were used to infect skinmuscle cells. Epimastigotes transformed into metacyclic trypomastigotes before entering host cells, multiplied, and completed the intracellular life cycle. We conclude that the amastigotes cultured in F-69 at 37 C are biologically similar to intracellular amastigotes from the vertebrate host, in that both can multiply and complete the life cycle intracellulary.     

Down-regulation of interleukin 1 (IL 1) receptor expression by IL 1 and fate of internalized 125I-labeled IL 1 beta in a human large granular lymphocyte cell line

The regulation of interleukin 1 (IL 1) receptor expression on a human large granular lymphocyte cell line, YT, and fate of internalized 125I-labeled IL 1 beta (125I-IL 1 beta) were studied. YT cells were selected for this study, because this cell line expresses a large number of specific high-affinity receptor for IL 1, responds biologically to exogenously added IL 1 by expressing high-affinity IL 2 receptors, and does not produce IL 1. YT cells constitutively express approximately 7 X 10(3) IL 1 receptors/cell with a Kd approximately 10(-10) M. Neither IL 2, phorbol myristic acid, nor lipopolysaccharide affected the total binding of 125I-IL 1 beta by YT cells. In contrast, the capacity of YT cells to bind 125I-IL 1 beta when incubated at 37 degrees C for 3 to 16 hr with a low dose of purified IL 1 beta (approximately 6 U/ml) was reduced by greater than 80%. The loss of binding capability gradually recovered by 16 hr after removal of IL 1 beta from cultured YT cells. The apparent loss of IL 1 receptor expression was accompanied by the internalization of 125I-IL 1 beta into cells. Acid treatment of YT cells to remove bound 125I-IL 1 beta at 4 degrees C showed that 50% of the 125I-IL 1 beta bound to cells could no longer be recovered after 30 min at 37 degrees C, and this increased to 80% after 3 hr at 37 degrees C. Fractionation of cell extracts on Percoll gradient additionally showed 125I-IL 1 beta to appear intracellularly after receptor binding on plasma membranes, and to be successively transferred to some membranous organelles (d approximately equal to 1.037) through an intermediate density organelle (d approximately equal to 1.050), and to finally end up in lysosomal cell fractions (d approximately equal to 1.05 to 1.08) after approximately 3 hr at 37 degrees C. Only approximately 5% of internalized 125I-IL 1 beta was released into culture media by 6 hr of incubation at 37 degrees C. However, the radioactivity in the TCA soluble fraction of the culture media increased gradually by 6 hr and a lysosomotropic enzyme, ethylamine, significantly inhibited both the transfer of internalized 125I-IL 1 beta to the lysosomal fraction and the degradation of 125I-IL 1 beta. This study represents the first evidence of autoregulation of IL 1 receptors by IL 1 and internalization of IL 1 molecules after binding to receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Opposing effects of dexamethasone on the clonal growth of granulocyte and macrophage progenitor cells and on the phagocytic capability of mononuclear phagocytes at different stages of differentiation

Dexamethasone, a synthetic glucocorticosteroid, was shown to modulate the colony-stimulating factor-dependent clonal growth of myeloid progenitor cells in semisolid agar cultures, enhancing the formation of granulocyte colonies (50–100%) and suppressing the formation of macrophage colonies (75–97%). Modulation of the pattern of myeloid colony formation by dexamethasone (12–125 nM) was brought about when the steroid was administered to 6-day cultures at the time of culture initiation and up to 72 hr later. Dexamethasone inhibited myeloid cell proliferation when administered to 5-day liquid cultures at culture initiation and up to 96 hr later. Dexamethasone (12–250 nM) also enhanced the phagocytic activity of bone marrow-derived mononuclear phagocytes toward heat-killed (HK) yeast cells (up to 100%) and IgG-coated sheep red blood cells (up to 60%). Enhancement of the phagocytic capability depended critically on the stage in culture at which dexamethasone was administered. Exposure to dexamethasone for 28 hr up to 96 hr of 96-hr cultures of bone marrow cells did not lead to a modulation of phagocytic activity of the developing mononuclear phagocytes. The presence of dexamethasone during the critical period of 96 hr to 120 hr after culture initiation led to an enhanced phagocytic capability, which was statistically significant already 12 hr after the administration of the glucocorticoid. Dexamethasone induced an enhanced phagocytic activity when administered at any time after culture initiation provided that it was in culture during this critical period. When added at 120 hr of culture, dexamethasone no longer enhanced the phagocytic capability of mononuclear phagocytes and when added later than 156 hr of culture suppressed it. Dexamethasone also suppressed (up to 68%) the phagocytic capability of resident and elicited peritoneal macrophages. The results suggest that glucocorticoids shift the balance of granulocyte vs. macrophage formation at early stages of precursor cell differentiation. Reduction in mononuclear phagocyte growth and enhancement of its phagocytic capability might reflect accelerated differentiation/maturation steps. The inhibitory effect of dexamethasone on macrophage formation and on the phagocytic capability of mature mononuclear phagocytes and peritoneal macrophages might be a relevant aspect of the in vivo immune suppression encountered after glucocorticoid administration.     

The effect of oxygen on the sporulation, δ-endotoxin synthesis and toxicity of Bacillus thuringiensis H14

Summary The sporulation and toxicity of Bacillus thuringiensis H14 were studied as a function of aeration. The fed-batch cultures carried out in the similar aeration conditions were followed in four different oxygen transfer rates containing 0, 20, 100 and 250 mmol/l/h. The percentage of total cells which had formed refractile spores in these four oxygen transfer rates were 100, 93, 84 and 48%, respectively. The highest rate of sporulation was observed in the absence of oxygen and the mature spores were the only population present under this condition at the end of culture. Sporulation in a large portion of cells failed under saturated oxygenation and either mature spores or vegetative cells were present at the end of culture. In the intermediate conditions, cells in different physiological states could be observed at the end of culture. It was found that the optimal conditions for spore yield and for δ-endotoxin yield were not the same, even though sporulation and δ-endotoxin formation proceed simultaneously during the fermentation process. The 130-kDa δ-endotoxin seemed to be more sensitive to aeration conditions. The higher toxicity against Culex pipiens was obtained under the saturated condition.     

Requirement of newly synthesized protein for the priming activity of interferon.

Pretreatment of mouse L cells with interferon (IF) enhanced IF production in response to polyinosinic-polycytidylic acid (poly I-poly C). Post-treatment of cells with IF caused no significant enhancement of IF production. The enhancing effect of IF pretreatment (priming) reached a maximum after incubation with IF (10 or 100 units/ml) for 4-6 hr at 37 C, but this effect was absent when the incubation was done at 4 C. Cells which were incubated for additional several hours at 37 C after IF pretreatment at 4 C did not develop the primed state nor the antiviral state. The presence of protein synthesis inhibitors during the IF pretreatment depressed, though not completely, the development of the primed state. The residual priming effect was lost when the cells were incubated with the inhibitors at 37 C for 2 hr before they were exposed to poly I-poly C. There was no significant difference in the binding rate of poly I-poly C to cells between IF-treated and untreated cells. The degradation rate of cell-bound poly I-poly C and its sensitivity to exogenous pancreatic ribonuclease in the pretreated cells were also similar to those in the untreated cells.     

Aeration time following ethylene oxide sterilization for reusable rigid sterilization containers: concentration of gaseous ethylene oxide in containers

Because ethylene oxide (EO) gas is toxic to humans, restrictions have been imposed on its use for sterilization, specifying allowable levels of residual EO remaining in sterilized apparatus and materials. However, the aeration time that optimizes the removal of the remaining EO when a rigid sterilizing container is used for a vessel had not been identified. Therefore, polyvinyl chloride, which easily adsorbs EO, was placed in rigid sterilizing containers, and aeration was carried out after 1, 8, 12, 17, and 24 hours. After standard EO sterilization, the EO concentrations remaining in the air in the rigid containers were measured. The results indicate that a period of 17 hours of aeration is appropriate when a rigid sterilizing container is used.     

Kinetics of CTL induction by concanavalin A.

Induction of maximal CTL activity was achieved within 12 hr of exposure to Con A in vitro in various mouse lymphoid cell populations. These included spleen cells from normal unsensitized mice, spleen cells from mice previously immunized with alloantigen, and mouse spleen cells exposed to alloantigen in long-term mixed leukocyte culture (LTMLC). Although induction of maximal incorporation of tritiated thymidine was accomplished within this same period in the cells obtained from LTMLC, a much longer period of Con A exposure (greater than 24 hr) was required for freshly prepared spleen cells from normal or previously immunized mice. These findings indicate that the increased tritiated thymidine uptake induced in freshly prepared spleen cells on continued exposure to Con A beyond 12 hr is not associated with the development of cytolytic activity, and that it probably represents stimulation of subpopulations no longer present in the LTMLC population where positive selection for cells responsive to cellular alloantigens has taken place.     

Epithelial sheet movement: effects of tunicamycin on migration and glycoprotein synthesis

Corneas with central epithelial wounds, 3 mm in diameter, were organ cultured in the presence of tunicamycin (TM) (1 microgram/ml), an antibiotic that inhibits glycosylation of asparagine-linked glycoproteins. Compared with control corneas, which healed in 22 hr, corneas cultured in the presence of TM for the entire culture time or for only the first 6 hr displayed a progressively slower epithelial healing rate that essentially dropped to zero by 24 hr of culture time. At 24 hr, approximately 75% of the wound was covered. After repeated washings with TM-free culture media (6X, 10 min each), this effect could consistently be reversed in corneas exposed to TM for 6 hr. Incorporation of [3H]glucosamine into trichloroacetic acid-precipitable proteins of migrating epithelial sheets was reduced to 14% that of controls after 12 hr of culture with TM, whereas [14C]leucine incorporation was not significantly affected. The decreased glycosylation was reflected on the cell surface after 12 and 20 hr culture in the presence of TM: apical cell membranes of the first six cells of the leading edge of the migrating sheet bound significantly fewer ferritin-concanavalin A particles per micrometer of membrane than did controls. These results indicate that synthesis of asparagine-linked glycoproteins is required for continued migration of corneal epithelial sheets. The asparagine-linked glycoproteins that are required for migration probably include cell-surface glycoproteins.