Development of Aspergillus parasiticus and formation of aflatoxin B1 under the influence of conidiogenesis affecting compounds

The influence of various inhibitors of hyphal growth, sporulation and biosynthesis of aflatoxin B₁ in Aspergillus parasiticus NRRL 2999 was studied. 6-Thioguanine, dl-ethionine, fluoroacetic acid and phenylboric acid, inhibitors of maturation of fungal conidiophores and of conidiogenesis, were added at various concentrations to malt extract agar. Lower concentrations of 6-thioguanine and dl-ethionine did not inhibit the growth of hyphae and the sporulation. Phenylboric acid reduced conidiogenesis more than hyphal growth. The yields of aflatoxin B₁ were significantly reduced. Additions of fluoroacetic acid did not greatly affect the growth of hyphae but totally inhibited the production of conidia and concurrently significantly reduced the formation of aflatoxin B₁. An interrelation between conidiogenesis and onset of secondary metabolism in A. parasiticus is evident.     

Regulation of biosynthesis of thiolutin and aureothricin inStreptomyces kasugaensis

l-Methionine anddl-ethionine decreased production of thiolutin and aureothricin inStreptomyces kasugaensis. In the presence ofl-methionine the culture also produced 3-methylthioacrylic acid, 3-methylthiopropionic acid and 3,6-bis-(2-methylthioethyl)-2,5-dioxopiperazine. Production of the metabolites depended on the concentration ofl-methionine in the medium.     

Morphology,cell-cell interactions,and migratory activity of IAR-2 epithelial cells transformed with the <Emphasis Type="Italic">RAS</Emphasis> oncogene: Contribution of cell adhesion protein E-Cadherin

The destruction of stable cell-cell adhesion and the acquisition of the ability to migrate are consistent stages of neoplastic evolution of tumor cells of epithelial origin. We studied the morphological and migration characteristics of epithelial cells of IAR1162 and IAR1170 clones derived from a mixed culture of N-RasV12 oncogene-transformed IAR-2 cell line. It was found that the oncogenic RAS can cause two types of morphological changes in IAR-2 epithelial cells. Cells of one type (IAR1162 clones) underwent epithelial-mesenchymal transition: they stopped to express E-cadherin, acquired fibroblast-like morphology, and did not form tight junctions. Cells of the other type (IAR1170 clones) retained a morphology close to the morphology of nontransformed progenitor cells, assembled E-cadherin-based adherens junctions and tight junctions, and formed a monolayer in confluent culture. However, in both IAR1162 and IAR1170 cells, the oncogenic RAS caused the destruction of marginal actin bundle and the reorganization of cell-cell adherens junctions. RAS-transformed IAR1162 and IAR1170 epithelial cells acquired the ability to migrate on a flat substrate as well as through narrow pores in membranes of migration chambers. A videomicroscopic study of transformed epithelial cell cultures demonstrated the instability of cell-cell contacts and the independent nature of cell migration. IAR1170 epithelial cells, which had E-cadherin-based adherens junctions, were also able to move as a group (collective migration). 1162D3 cells, which lost the ability to express endogenous E-cadherin as a result of Ras-transformation, were transfected with a plasmid carrying the CDH1. As a result of transfection, clones of cells with different levels of expression of exogenous E-cadherin were obtained. The high level of expression of exogenous E-cadherin in transformed epithelial cells led to a decrease in the rate of migration on a two-dimensional substrate of the cells that were in contact with neighboring cells but almost had no effect on the migration of single cells, at the same time increasing the number of cells that migrated through the pores in migration chambers. Thus, the destruction of marginal actin bundle and the change in the spatial organization of cell-cell adherens junctions, irrespective of the presence or absence of E-cadherin, was accompanied by destruction of stable cell-cell adhesion and the appearance of cell motility in Ras-transformed epithelial cells. The retaining of E-cadherin in cell-cell adhesion junctions affects the motility of transformed epithelial cells and plays an important role in their collective migration.     

Disorders of the actin cytoskeleton in transformed epithelial cells

The actin cytoskeleton of 8 transformed epithelial cell lines was studied using electron microscopy of platinum replicas. Seven of these lines belonged to the IAR series of rat liver epithelial cells, being at different stages of neoplastic progression. One cell line (FBT) was derived from the epithelium of bovine fetal trachea. The extent of actin cytoskeleton alteration in cell lines studied has been shown to correlate with other signs of neoplastic transformation. Among various actin-containing cell structures (microfilament bundles, actin meshwork at active edges, cell-cell adherence junctions, and endoplasmic microfilament sheath) the latter was the most sensitive to transformation. The loosening of the sheath and the alteration of its fine structure were observed in all the cell lines. The degree of these changes increased in the following order: FBT; non-tumorigenic IAR lines; IAR lines transformed in vitro; IAR lines obtained from the latter by single or double selection in vivo. The alteration of sheath was the only disturbance of actin cytoskeleton in FBT cells, whereas in other groups of epithelial cell lines some other changes occurred. These involved disruption of actin-containing intercellular junctions, the cell polarization accompanied by progressive shortening of length of the cell active edge containing actin meshwork, and disappearance or reorganization of microfilament bundles.     

Membrane formation and membrane contacts during the development of the sea urchin embryo

Summary Sea urchin embryos, 8-cell stage to pluteus stage, fixed in osmium tetroxide and embedded in Epon 812 were observed by electron microscopy. At no point in the development were syncytial junctions between the embryonic cells found. During the cleavage stages the membrane contact was closer than in later stages. In early blastula stages intercellular clefts appeared which in the gastrula stage demarcate every cell. At the same time a ringshaped desmosome structure develops at the outer cell surface. In the pluteus stage a closer cell contact is re-established. With proceeding embryogenesis endoplasmic membranes will attach to the cell membrane. These membrane structures may even be of nuclear origin. Gradually, long protrusions, vesicles and lamellae begin to be formed from the nuclear membrane. The commencement of this nuclear activity coincides in time with the formation of nucleoli. At cell division the new cell membrane seemed to arise partly independently of the cleavage furrow from a system of cytoplasmic vesicles.The investigation was facilitated by grants from the Nordic Insulin Foundation.I am indebted to Dr. Torsten Olsson and Miss Brita Nilsson for procuring the material and to Mrs. Mariann Carleson for technical aid.     

Rho-stimulated Contractility Contributes to the Fibroblastic Phenotype of Ras-transformed Epithelial Cells

Oncogenic transformation of cells alters their morphology, cytoskeletal organization, and adhesive interactions. When the mammary epithelial cell line MCF10A is transformed by activated H-Ras, the cells display a mesenchymal/fibroblastic morphology with decreased cell–cell junctions but increased focal adhesions and stress fibers. We have investigated whether the transformed phenotype is due to Rho activation. The Ras-transformed MCF10A cells have elevated levels of myosin light chain phosphorylation and are more contractile than their normal counterparts, consistent with the activation of Rho. Furthermore, inhibitors of contractility restore a more normal epithelial phenotype to the Ras-transformed MCF10A cells. However, inhibiting Rho by microinjection of C3 exotransferase or dominant negative RhoA only partially restores the normal phenotype, in that it fails to restore normal junctional organization. This result prompted us to examine the effect that inhibiting Rho would have on the junctions of normal MCF10A cells. We have found that inhibiting Rho by C3 microinjection leads to a disruption of E-cadherin cytoskeletal links in adherens junctions and blocks the assembly of new adherens junctions. The introduction of constitutively active Rho into normal MCF10A cells did not mimic the Ras-transformed phenotype. Thus, these results lead us to conclude that some, but not all, characteristics of Ras-transformed epithelial cells are due to activated Rho. Whereas Rho is needed for the assembly of adherens junctions, high levels of activated Rho in Ras-transformed cells contribute to their altered cytoskeletal organization. However, additional events triggered by Ras must also be required for the disruption of adherens junctions and the full development of the transformed epithelial phenotype.     

Optimal conditions for induction of certain mutation types inBrevibacterium flavum by N-Methyl-N’-nitro-N-nitrosoguanidine

Conditions under which it is possible to induce auxotrophic mutants and DL-selenomethionine-resistant mutants inB. flavum by N-methyl-N’-nitro-N-nitrosoguanidine were determined. The yield of auxotrophic mutants was increased to 3% during mutagenesis in the first stage and to 1.5% in the second stage when using the enrichment-selective method with vancomycin. The optimal vancomycin concentration for inactivation of prototrophic cells growing in a minimal medium was 200 mg/L and the optimal time of treatment was 8 h. When testing the effect of three amino acid analogues (dl-ethionine,dl-selenomethionine and L-methionine sulfoximine) it was found thatB. flavum is sensitive todl-selenomethionine present in the minimal cultivation medium. Mutants resistant to 1 mg/mL of selenomethionine were isolated. Both isotope studies and measurement of growth indicate thatdl-ethionine also entersB. flavum cells, although its competition with endogenously synthesized methionine is not significant.     

Morphology, cell-cell interactions, and migratory activity of IAR-2 epithelial cells transformed with the RAS oncogene: contribution of cell adhesion protein E-cadherin

The destruction of stable cell-cell adhesion and the acquisition of the ability to migrate are consistent stages of neoplastic evolution of tumor cells of epithelial origin. We studied the morphologic and mi gration characteristics of epithelial cells of Iar1162 and IAR1170 clones derived from a mixed culture of on cogene N-RasV12-transformed cell line IAR-2. It was found that the mutant oncogene RAS can cause two types of morphological changes in IAR-2 epithelial cells. Cells of one type (IAR1162 clones) underwent epithelial-mesenchymal transition: they stopped to express E-cadherin, acquired fibroblast-like morphology, and did not form tight junctions. Cells of the other type (IAR1170 clones) retained a morphology close to the morphology of nontransformed progenitor cells, formed E-cadherin-based adherens junctions and tight junctions, and formed a monolayer in confluent culture. However, in both IAR1162 and IAR1170 cells, the mutant oncogene RAS caused the destruction of marginal actin bundle and the reorganization of cell-cell adherens junctions. RAS-transformed IAR1162 and IAR1170 epithelial cells acquired the ability to migrate on a flat substrate as well as through narrow pores in membranes of migration chambers. A videomicroscopic study of transformed epithelial cell cultures demonstrated the instability of cell-cell contacts and the independent nature of cell migration. IAR 1170 epithelial cells, which had E-cadherin-based adherens junctions, were also able to move as a group (collective migration). 1162D3 cells, which lost the ability to express endogenous E-cadherin as a result of Ras-transformation, were transfected with a plasmid carrying the CDH1. As a result of transfection, clones of cells with different levels of expression of exogenous E-cadherin were obtained. The high level of expression of exogenous E-cadherin in transformed epithelial cells led to a decrease in the rate of migration on a two-dimensional substrate of the cells that were in contact with neighboring cells but almost had no effect on the migration of single cells, at the same time increasing the number of cells that migrated through the pores in migration chambers. Thus, the destruction of marginal actin bundle and the change in the spatial organization of cell-cell adherens junctions, irrespective of the presence or absence of E-cadherin, was accompanied by destruction of stable cell-cell adhesion and the appearance of locomotor activity in Ras-transformed epithelial cells. The retaining of E-cadherin in cell-cell adhesion junctions affects the locomotor activity of transformed epithelial cells and plays an important role in their collective migration.     

Properties of Wilms' tumor line (TuWi) and pig kidney line (LLC-PK1) typical of normal kidney tubular epithelium

Summary Two stable epithelial-like cell lines, the pig kidney strain (LLC-PK₁) and a Wilms' tumor line (TuWi), previously established in other laboratories, were found to exhibit a number of properties characteristic of kidney proximal tubular epithelium. Electron micrographs of LLC-PK₁ monolayers revealed cells forming rosettes reminiscent of tubules. Numerous elongated microvilli and an amorphous basal laminar material surrounded the cell membranes. Cell junctions were located between cell membranes at regions adjacent to the patent lumens. Wilms' cells in culture were similar in appearance to the pig kidney cells; they exhibited numerous microvilli, a thin basal laminar coating on the membrane, and desmonsomes between cells. No rosette formation was evident. Neither cell line was found to produce extracellular reticulin fibers when grown in the presence ofl-ascorbic acid for 1 week. Absence of stainable reticulin in cell monolayer culture after ascorbicacid treatment has been noted only in cell lines of apparent epithelial origin. Histochemically, both lines reacted positively for activities of a number of enzymes found in high amounts in normal kidney tubular epithelium. Pig kidney cells were highly positive for γ-glutamyl transpeptidase activity and moderately active for acid phosphatase and leucine aminopeptidase activities. Wilms' tumor cells were markedly active for γ-glutamyl transpeptidase, 5′-nucleotidase, ATPase, glucose-6-phosphatase, and acid phosphatase activities. These findings in conjunction with the ultrastructural observations indicate that these two lines in culture maintain many of the properties typical of proximal kidney tubular epithelium.     

Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line

In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.     

Expression of the Fusarium oxysporum lactonase gene in Aspergillus oryzae: molecular properties of the recombinant enzyme and its application

The lactonase gene of Fusarium oxysporum was expressed in Aspergillus oryzae for optical resolution of dl-pantoyl lactone. When the chromosomal gene encoding the full-length form of the lactonase, which has its own NH₂-terminal signal peptide, was introduced in the host cells, the resulting transformant produced an enzyme of 46,600 Da, which corresponded to the wild-type enzyme. In contrast, A. oryzae transformed with the cDNA coding the mature enzyme produced a protein of 41,300 Da. Deglycosylation analysis with an endoglycosidase revealed that the difference in molecular mass arose from the different sugar contents of the recombinant enzymes. The mycelia of the transformant were used as a catalyst for asymmetric hydrolysis of dl-pantoyl lactone. The initial velocity of the asymmetric hydrolysis reaction catalyzed by the transformant was estimated to be 30 times higher than that by F. oxysporum. When the mycelia of the transformant were incubated with a 20% dl-pantoyl lactone solution for 4 h, 49.9% of the racemic mixture was converted to d-pantoic acid (>95% ee).     

Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras

The effects of viral Kirsten ras oncogene expression on the polarized phenotype of MDCK cells were investigated. Stable transformed MDCK cell lines expressing the v-K-ras oncogene were generated via infection with a helper-independent retroviral vector construct. When grown on plastic substrata, transformed cells formed continuous monolayers with epithelial-like morphology. However, on permeable filter supports where normal cells form highly polarized monolayers, transformed MDCK cells detached from the substratum and developed multilayers. Morphological analysis of the multilayers revealed that oncogene expression perturbed the polarized organization of MDCK cells such that the transformed cells lacked an apical--basal axis around which the cytoplasm is normally organized. Evidence for selective disruption of apical membrane polarity was provided by immunolocalization of membrane proteins; a normally apical 114-kD protein was randomly distributed on the cell surface in the transformed cell line, whereas normally basolateral proteins remained exclusively localized to areas of cell contact and did not appear on the free cell surface. The discrete distribution of the tight junction-associated ZO-1 protein as well as transepithelial resistance and flux measurements suggested that tight junctions were also assembled. These findings indicate that v-K-ras transformation alters cell-substratum and cell-cell interactions in MDCK cells. Furthermore, v-K-ras expression perturbs apical polarization but does not interfere with the development of a basolateral domain, suggesting that apical and basolateral polarity in epithelial cells may be regulated independently.     

Drug-metabolizing enzyme activities in freshly isolated oval cells and in an established oval cell line from carcinogen-fed rats

The activities of several different phase I and phase II drug-metabolizing enzymes were measured in freshly isolated oval cells from rats fed a choline-deficient/DL-ethionine-supplemented diet for 6 weeks and alsoin vitro in the established oval cell line OC/CDE 6. No cytochrome P450 was spectrophotometrically measurable in both preparations and two cytochrome P450-dependent monoxygenase activities, aminopyrineN-demethylase and ethoxyresorufinO-deethylase, could not be detected in the oval cells of both sources. However, cytosolic glutathione transferase, microsomal expoxide hydrolase and UDP-glucuronosyltransferase activities were clearly measurable in oval cells. Similar enzyme activities were found in freshly isolated and cultured oval cells. The highest activities of these three enzymes were detected during the exponential growth phase of the cultured cells; thereafter the activities decreased until the cells reached confluency. Changes in phenol UDP-glucuronosyltransferase (UGT1A1) mRNA levels paralleled the variations in UDP-glucuronosyltransferase activity, i.e. they were high in exponentially growing oval cells and low in confluent cell cultures. Taking into account that oval cells are able to proliferate in the livers of rats continuously fed a choline-deficient/DL-ethionine-supplemented diet and that none of the analyzed drug metabolizing enzymes are involved in the activation or detoxication ofDL-ethionine, the described pattern might be part of a more general, nonspecific, protection mechanism enabling these cells to overcome the cytotoxic effects of a variety of carcinogens and to proliferate even in their presence. Furthermore, the expression of microsomal epoxide hydrolase, cytosolic glutathione transferase and UDP-glucuronosyltransferase appears to depend on the proliferative status of the cells.Abbreviations CDE choline-deficient/DL-ethionine-supplemented diet - GST glutathione transferase - mEH microsomal epoxide hydrolase - UGT UDP-glucuronosyltransferase     

Mouse mammary cells ind-valine medium

Summary Cells of a mouse mammary epithelial cell line as well as fibroblasts from a mouse mammary explant were severely inhibited from proliferating in a medium in whichd-valine was substituted forl-valine. After the first few days ind-valine medium, the number of epithelial cells did not increase despite the fact that a few percent continued to synthesize DNA. The cells did recognize the presence of thed-valine in the medium because cells ind-valine increased in volume and their numbers remained stationary, whereas cells without valine shrank and the cell numbers decreased with time. The NMuMG cells were obtained from Mr. Robert Owens and were produced with support from the National Cancer Institute, Biological Carcinogenesis Branch, Division of Cancer Cause and Prevention under the auspices of the Office of Naval Research and the Regents of the University of California. This project was funded by the National Cancer Institute, Bethesda, MD, Contract N01-CB-74094.     

Characterization of prolidase I and II purified from normal human erythrocytes: comparison with prolidase in erythrocytes from a patient with prolidase deficiency

The effect of various sulfur-containing amino acids on the activities of prolidase isoenzymes I and II isolated from erythrocytes of healthy individuals, and erythrocyte lysates from a patient with prolidase deficiency was investigated. The activity of prolidase I against glycylproline was strongly enhanced by d-methionine. l-Methionine and d,l-methionine slightly enhanced the activity at low concentration, but N-acetyl-l-methionine had no effect. d-Ethionine, l-ethionine, and d,l-ethionine also enhanced the activity of prolidase I. d,l-Homocysteine enhanced the activity at low concentration, but inhibited the activity at 50 mM. The activity of prolidase II against methionylproline was enhanced by d-methionine, d,l-methionine, and l-methionine, but N-acetyl-l-methionine had no effect. d-Ethionine and d,l-ethionine strongly enhanced the activity of prolidase II compared with l-ethionine; d,l-homocysteine weakly enhanced the activity. d,l-Homocysteine-thiolactone inhibited the activities of prolidase I and II in a concentration-dependent manner. The effect of various sulfur-containing amino acids on prolidase activity against methionylproline in erythrocyte lysates from a patient with prolidase deficiency was almost the same as that on prolidase II. The kinetics of the activities of prolidase I, II, and patient prolidase were also studied. Their K _m values were changed by adding sulfur-containing amino acids, but V _max values were unchanged.     

Ultrastructural and biological characterization of human choroid cell cultures transformed by Simian Virus 40

Summary A human diploid cell line of choroid origin was isolated from the retrouveal portion of an enucleated eye and designated HC. After 10 passages, when the proliferative capacity of HC cells decreased, they were infected and transformed by Simian Virus 40 (SV40). A proliferating long-term cultured cell line designated HC/SV40 was established and it has been maintained as monolayer for more than 100 passages so far. The two cell lines, HC and HC/SV40, were compared for growth characteristics, capacity to form colonies in soft agar, presence of nuclear T-antigen, and ultrastructure. Cytogenetic analysis was also performed to determine the presence of chromosomal aberrations due to the permanent viral transformation of the cell line. The results indicate that HC/SV40 should be considered the transformed counterparts of HC cells because they are morphologically similar to the latter but can grow in soft agar, possess T-antigen, and show a pattern of karyotypic changes similar to that induced by SV40 in human fibroblasts. The choroid origin of HC and HC/SV40 cell lines was confirmed by the presence, in their cytoplasm, of typical electron dense granules. Their neural origin will make these cell lines very useful for neuropharmacological and differentiation studies. This work was supported by Grant 82.00346.96 from the C.N.R. finalized project “Control of Neoplastic Growth”.     

Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells

Cells dissociated from normal prelactating mouse mammary glands or from spontaneous mammary adenocarcinomas, when grown at high density on an impermeable substrate, form nonproliferating, confluent, epithelial pavements in which turgid, blister-like domes appear as a result of fluid accumulation beneath the cell layer. To compare the structure of the fluid-segregating cell associations in normal and tumor cell cultures with that of lactating gland in vivo, we have examined such cultures alive and in thick and thin sections and freeze-fracture replicas. Pavement cells in all cases are polarized toward the bulk medium as a lumen equivalent, with microvilli and continuous, well-developed occluding junctions at this surface. Between the pavement and the substrate are other cells, of parenchymal or stromal origin, scattered or in loose piles; these sequestered cells are relatively unpolarized and never possess occluding junctions. Small gap junctions have been found in the pavement layer, and desmosomes may link epithelial cells in any location. Under the culture conditions used, development of the epithelial secretory apparatus is not demonstrable; normal and neoplastic cells do not differ consistently in any property examined. A dome's roof is merely a raised part of the epithelial pavement and does not differ from the latter in either cell or junction structure. We suggest that dome formation demonstrates the persistence of some transport functions and of the capacity to form effective occluding junctions. These basic epithelial properties can survive both neoplastic transformation and transition to culture.     

Efficient biocatalytic production of <Emphasis Type="SmallCaps">d</Emphasis>-4-hydroxyphenylglycine by whole cells of recombinant <Emphasis Type="Italic">Ralstonia pickettii</Emphasis>

The first establishment of a homologous expression system in the host Ralstonia pickettii CGMCC1596 using the compatible broad-host-range plasmid pWB5 is described. When whole cells of the recombinant strain R. pickettii MMYY01 (CGMCC1596/pYY05) were used as the biocatalyst to transform dl-4-hydroxyphenylhydantoin (dl-HPH) to d-4-hydroxyphenylglycine (d-HPG), the conversion rate reached 94 % in first 9 h, at a production rate of 2.8 g L⁻¹ h⁻¹, with the rapid reduction of the intermediate [N-carbamoyl-2-(4-hydroxyphenyl)glycine], compared with 80 % in >50 h at a rate of 0.5 g L⁻¹ h⁻¹ for the CGMCC1596. The stability of the recombinant plasmid pYY05 is sufficient for its application in industrial batch fermentation. An alternative strategy for the conversion of dl-HPH to d-HPG by resting CGMCC1596 cells and heterologous DCase expressed by E. coli is discussed.     

Different locations of carbohydrate-containing sites in the surface membrane of normal and transformed mammalian cells

Summary A soybean agglutinin was found to agglutinate mouse, rat and human cell lines transformed by viral carcinogens, but not hamster cells transformed by viral or non-viral carcinogens. Normal cells from which the transformed cells were derived were not agglutinated by this agglutinin, but they were rendered agglutinable after short incubation with trypsin or pronase. The transformed hamster cells, on the other hand, became agglutinable only after prolonged treatment with pronase. The agglutination was specifically inhibited by N-acetyl-d-galactosamine, indicating that N-acetyl-d-galactosamine-like saccharides are part of the receptor sites for soybean agglutinin on the surface membrane. Such sites exist in a cryptic form in normal cells; they are exposed in transformed mouse, rat and human cells, but become less accessible in transformed hamster cells. The receptor sites for soybean agglutinin differ from the receptors for two other plant agglutinins (wheat germ agglutinin that interacts with N-acetyl-d-glucosamine-like sites and Concanavalin A that interacts with -d-glucopyranoside-like sites) which become exposed upon transformation of all lines tested. In normal hamster cells, the receptors for all three agglutinins become exposed after incubation with trypsin, but the exposure of N-acetyl-d-galactosamine-like sites requires the longest enzyme treatment. The results indicate a difference in the location of different carbohydrate-containing sites in the surface membrane. The differences in the exposure of carbohydrate-containing sites in the membrane could not be correlated with the levels of carbohydrate-splitting glycosidases in normal and transformed cells.