Osteogenic and osteoclastic cell interaction: development of a co-culture system

The processes involved in the regulation of bone cell metabolism are complex, including those implicated in bone cell coupling. This study was undertaken to develop a model that would permit real-time interaction between osteoclastic cells and osteoblasts in vitro. Osteogenic bone marrow stromal cells were isolated from 18-day-old embryonic chickens, while osteoclastic cells were isolated from laying White Leghorn hens on calcium-deficient diets. Osteoclastic cells (5×10⁵) were seeded onto mineral thin films and suspended above osteogenic cells (1×10⁴) already plated on the bottoms of tissue culture plate wells. The data showed that after 4 days of incubation there was up to a fivefold (P<0.05) reduction in all measured parameters of osteogenesis (mineralization, alkaline phosphatase activity and type I collagen production) in osteogenic cultures grown in the presence of osteoclastic cells. Similarly, osteoclastic cell-induced mineral resorption was reduced up to threefold (P<0.05). Co-culture effects on cellular responses could be manipulated by known antiresorptive agents (e.g., pamidronate) altering either the source or the age of osteoclastic cells. The results indicate that the co-culture model may be useful in the study of bone cell interactions.     

An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis

A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded in a thin layer of alginate and are nourished from the medium in the underlying basislayer. In the alginate layer the protoplasts regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l⁻¹ kanamycin or 5 mg l⁻¹ paromomycin. Single resistant cells can be recovered from about 10 000 sensitive cells in one alginate layer. Injection of theneo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian fashion.     

Thin layer gel filtration as a method for routine steroid receptor binding studies

A new technique is introduced which allows the quantitative determination of radioactively labeled substances from thin layer gel filtration (TLG). For gel filtration sephadex G-100 on a glass plate coated with a cellulose film was used. Together with the Sephadex layer the celluose film could be cut easily into strips and placed into vials for counting. The method was satisfactory with binding studies of steroid hormones on the prostatic androgen receptor. Because of simplicity, speed, and economical reasons this method could be used as a routine receptor assay.The binding constant of dihydrotestosterone (DHT) receptor complex was determined as K_a = 0.36 × 10⁹ liter/mole. The relative binding affinity of the antiandrogen cyproterone acetate was found to be 0.23.     

Morphological Transitions Governed by Density Dependence and Lipoxygenase Activity in Aspergillus flavus

Aspergillus flavus differentiates to produce asexual dispersing spores (conidia) or overwintering survival structures called sclerotia. Results described here show that these two processes are oppositely regulated by density-dependent mechanisms and that increasing the cell density (from 10¹ to 10⁷ cells/plate) results in the lowest numbers of sclerotial and the highest numbers of conidial. Extract from spent medium of low-cell-density cultures induced a high-sclerotium-number phenotype, whereas high-cell-density extract increased conidiation. Density-dependent development is also modified by changes in lipid availability. Exogenous linoleic acid increased sclerotial production at intermediate cell densities (10⁴ and 10⁵ cells/plate), whereas oleic and linolenic acids inhibited sclerotium formation. Deletion of Aflox encoding a lipoxygenase (LOX) greatly diminished density-dependent development of both sclerotia and conidia, resulting in an overall increase in the number of sclerotia and a decrease in the number of conidia at high cell densities (>10⁵ cells/plate). Aflox mutants showed decreased linoleic acid LOX activity. Taken together, these results suggest that there is a quorum-sensing mechanism in which a factor(s) produced in dense cultures, perhaps a LOX-derived metabolite, activates conidium formation, while a factor(s) produced in low-density cultures stimulates sclerotium formation.     

Dynamic studies of proton diffusion in mesoscopic heterogeneous matrix: II. The interbilayer space between phospholipid membranes

The thin water layer, as found in chloroplast or mitochondria, is confined between low dielectric amphypathic surfaces a few nm apart.

The physical properties of this mesoscopic space, and how its dimensions affect the rate of chemical reactions proceeding in it, is the subject for this study.

The method selected for this purpose is time resolved fluorometry which can monitor the reversible dissociation of a proton from excited molecule of pyranine (8 hydroxy pyrene 1,3,6 tri sulfonate) trapped in thin water layers of a multilamellar vesicle made of neutral or slightly charged phospholipids.

The results were analyzed by a computer program of N. Agmon (Pines, E., D. Huppert, and N. Agmon. 1988. J. Am. Chem. Soc. 88:5620-5630) that simulates the diffusion of a proton, subjected to electrostatic attraction, in a thin water layer enclosed between low affinity, proton binding surfaces. The analysis determines the diffusion coefficient of the proton, the effective dielectric constant of the water and the water accessibility of the phosphomoieties of the lipids.

These parameters were measured for various lipids [egg-phosphatidylcholine (egg PC), dipalmitoyl phosphatidylcholine (DPPC), cholesterol + DPPC (1:1) and egg PC plus phosphatidyl serine (9:1)] and under varying osmotic pressure which reduces the width of the water layer down to ~10 ~ across.

We found that: (a) The effective dielectric constant of the aqueous layer, depending on the lipid composition, is ~40. (b) The diffusion coefficient of the proton in the thin layer (30-10 ~ across) is that measured in bulk water D = 9.3 10^-5 cm²/s, indicating that the water retains its normal liquid state even on contact with the membrane. (c) The reactivity of the phosphomoiety, quantitated by rate of its reaction with proton, diminishes under lateral pressure which reduces the surface area per lipid.

We find no evidence for abnormal dynamics of proton transfer at the lipid water interface which, by any mechanism, accelerates its diffusion.

     

A fluorometric assay for the quantitation of aminosugars.

A method is presented for the quantitative determination of aminosugars in glycoproteins. Glycoproteins are acid hydrolyzed and reacted with the fluorogenic reagent 1-dimethylaminonaphthalene-5-sulfonyl chloride. Following cellulose thin layer electrophoresis in pyridine/acetic acid (pH 4.4), 5 h at 500 V, appropriate areas of the plate are scraped and the dansyl hexosamines are eluted with 95% ethanol. Aliquots are then applied to a silica gel tlc plate and subjected to ascending thin layer chromatography in cyclohexane/ethylacetate/ethanol (6/4/3). After spraying the plate with triethanolamine/isopropanol ($\text{1}\text{4}$), fluorescent intensities are measured by in situ scanning. The aminosugar content of the glycoprotein is determined from a curve generated from a series of standards run concurrently on each plate. The method clearly resolves glucosamine from galactosamine, and is sensitive for the detection of aminosugars in the subnanomole range.     

Preparation of a cell-free translation system from PC12 cells

The postmitochondrial fraction (S10) contains the cellular components essential for translation, and a high-salt wash (HSW) of the ribosomes is enriched in eukaryotic initiation factors. This report describes the preparation of a cell-free translation system utilizing an S10 extract from PC12 cells. The products synthesized from either firefly luciferase mRNA or PC12 cell poly(A) RNAs in the PC12-S10 extract were increased by the addition of the HSW from PC12 cells. Increases in the translation of luciferase mRNA by the addition of PC12-HSW were dose-dependent and also dependent on the time of incubation. The translation of human epidermal growth factor receptor (hEGFR) mRNA could also be detected in the PC12-S10 extract translation system by immunoprecipitation.N-linked glycosylation of the translation products also was observed. The efficiency of translation was altered by the addition of Mg²⁺ or K⁺, and optimization of the concentrations of these ions was necessary for each mRNA. The translation system made from PC12 cells, then, is capable of the synthesis of proteins of relatively high molecular weight and should be useful for analyzing mechanisms of translational control during proliferation and differentiation of cells from a neuronal lineage. Special issue dedicated to Dr. Hans Thoenen.     

High Frequency of Natural Genetic Transformation of Pseudomonas stutzeri in Soil Extract Supplemented with a Carbon/Energy and Phosphorus Source

Agar medium (SME) prepared from aqueous soil extract was used to examine genetic transformation of Pseudomonas stutzeri JM302 (his-1) by homologous his⁺ DNA in a plate transformation assay. Growth studies indicated that SME was strongly limited in carbon and nitrogen sources. Transformation was observed on SME supplemented with pyruvate, phosphate, and ammonium. A 25-fold increase of the transformation frequency was obtained with nitrogen limitation when SME was supplemented with only pyruvate plus phosphate. Similar results were obtained with artificial soil extract medium prepared on the basis of the chemical analysis of the soil extract. On a standard minimal medium, transformation frequencies also increased (10- to 60-fold) when ammonium, phosphate, or pyruvate was growth limiting. Limitation of two or three nutrients did not stimulate transformation. The size of the inoculum (2 × 10³ to 2 × 10⁷ cells) was irrelevant to the enhanced transformation under nitrogen limitation on SME or standard minimal medium. We further show that P. stutzeri can use a variety of carbon and energy sources for competence development. It is concluded that genetic transformation of P. stutzeri is possible in the chemical environment of soil upon supply of nutrients and may be strongly stimulated by a growth-limiting concentration of single nutrients including sources of C, N, or P.     

Characterization of an in vitro proliferation response to solubilized Trichinella spiralis antigens: Role of Ia antigens and Ly-1+ T cells

An antigen extract from Trichinella spiralis muscle larvae was prepared and used to immunize strains of mice which were either relatively resistant (B10.S) or susceptible (B10.BR) to oral infection with T. spiralis larvae. Proliferation of cells from the draining lymph nodes was then measured in vitro after stimulation with the T. spiralis extract as well as appropriate control antigens. Primed cells from resistant B10.S mice responded better to challenge than did cells from the susceptible B10.BR strain. Cell-depletion experiments involving B10.S cells indicated that the in vitro cell proliferation response is dependent upon Ly-1⁺ T cells. The data were also consistent with a requirement for Ly-1⁺, -2⁺, and -3⁺ amplifier cells. Administration of anti-I^s serum to the cultures specifically inhibited (nearly 75%) the cell proliferation response. The potential applications of this system as a tool in immunogenetic analyses of immunity to T. spiralis are discussed.     

Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium

Summary An improved procedure has been developed for clonal growth of normal human epidermal keratinocytes (HK) without feeder cells or conditioned medium. The use of medium 199, supplemented with 0.4 μg/ml hydrocortisone (HC) and 20% (v/v) whole fetal bovine serum (wFBS) and conditioned overnight by 3T3 cells, eliminated the need for a feeder layer of lethally irradiated 3T3 cells for HK growth. Several other media with equivalent conditioning and supplementation failed to support satisfactory multiplication of HK, including Dulbecco's modified Eagle's medium, which is normally used for growth of HK with a feeder layer. Increasing the concentration of HC to 10 μg/ml (2.8×10⁻⁵ M) made possible clonal growth of HK without any conditioning of the medium. The addition of 10⁻⁵ M putrescine, 10⁻⁵ M vitamin B₁₂, or 3.7×10⁻⁶ M β-estradiol further enhanced growth in unconditioned medium. Substantially greater improvement was obtained by the addition of pituitary extract or fractions prepared from pituitary extract. In medium 199 supplemented with 10 μg/ml HC, 20% (v/v) wFBS, and 0.15 mg/ml each of two pituitary fractions, single HK attach with a colony-forming efficiency equal to that in conditioned medium and form stratified, keratinized colonies that grow to confluency and can be subcultured. These results make it clear that HK do not require special “conditioning factors” from fibroblasts for clonal growth and differentiation in culture. Thus, factors directly involved in growth and the expression of differentiation can be analyzed without the interfering effects of any other type of cell. Preliminary studies with epidermal growth factor (EGF), which stimulates growth and extends life span of HK grown in the presence of fibroblasts, have shown that, in the absence of fibroblasts, EGF has no effect either on clonal growth or on cumulative multiplication potential of HK. This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by Donna M. Peehl in partial fulfillment of the requirements for the Ph.D. degree. This work was supported by Grant CA 15305 from the National Cancer Institute and Grant AG 00310 from the National Institute on Aging.     

A rapid and sensitive fluorescence assay for angiotensin-converting enzyme.

A new, highly sensitive, specific assay for dopamine-β-hydroxylase (DBH) activity in human serum is described. Tyramine is used as a substrate; the product of the enzymatic hydroxylation, octopamine, is converted by reacting with 1-dimethylaminonaphthalene-5-sulfonyl-chloride (Dns-Cl) to a fluorescent product, which is extracted from the reaction mixture and purified from the extract by thin-layer chromatography (tlc). The fluorescence of the dansylated octopamine is measured in situ on the tlc plate using a chromatogram-spectrofluorometer. This one-step enzyme reaction can be performed at optimum pH and substrate concentration. As little as 8 ng of octopamine can be determined accurately; the response is linear up to more than 400 ng of octopamine. A comparison with the radioenzymatic assay (Weinshilboum, R., and Axelrod, J. (1971) Circ. Res.28, 307–315) shows an approximately twofold increase in the enzymatic activity measured. Kinetic studies of human sera with high and low DBH activity gave a K_m value of 3.1 × 10^?3m. The method is successfully being used for the functional characterization of the enzyme and genetic studies (Herschel, M., in preparation).     

A radioenzymatic assay for catecholamines and dihydroxyphenylacetic acid.

Picogram quantities of the catecholamines, dopamine, norepinephrine, and epinephrine, and the dopamine metabolite, dihydroxyphenylacetic acid, can be measured in tissue or plasma samples utilizing a rapid radioenzymatic procedure. The catechols are converted to their ³H-methylated derivatives (3-methoxytyramine, normetanephrine, metanephrine and homovanillic acid, respectively) by the enzyme catechol-O-methyltransferase with ³H-S-adenosylmethionine serving as the ³H-methyl donor. Following the enzymatic reaction, unreacted ³H-S-Adenosylmethionine is removed by precipitation and the reaction products are separated by thin layer chromatography on silica plates. The areas corresponding to the ³H-methylated derivatives are scraped into scintillation vials, eluted with aqueous buffer, extracted into nonpolar scintillation cocktail, and counted by liquid scintillation spectrometry. Using the standard assay procedure described here, over 100 tubes can be assayed in a single day with a sensitivity of 15–25 pg for all compounds measured. With the application of additional procedures, as little as 1 pg norepinephrine and epinephrine and 5–10 pg dopamine and dihydroxyphenylacetic acid can be quantified in a single sample.     

Chromatographic performance of a thin microporous bed of nitrocellulose

Chromatography along thin (125 μm) porous beds of nitrocellulose, layered on top of an polyester backing, shows good separation efficiency with plate heights of 10–20 μm. Flow is controlled by capillary forces and shows low rate variations between the individual disposable devices. Positively charged groups were introduced into the nitrocellulose and efficient separation of transferrin isoforms, differing by only 0.1 pI units, was found after a short migration distance (1 cm). The upper surface is not covered, which allows sample and reagents to be added, and the clear backing permits detection. The chromatography can easily be combined on-line with sensitive immunoassay detection down to the pM (10⁻¹² M) range. This microscaled combination device should have a wide range of applications in analytical biochemistry.     

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined ¹. This is particularly true in autoimmune diseases and cancer². Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes ^1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays ⁶. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.     

A Thin‐Layer Liquid Culture Technique for the Growth of Helicobacter pylori

Background and Aims: Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin‐layer liquid culture technique for the growth of H. pylori. Methods: A thin‐layer liquid culture system was established by adding liquid media to a 90‐mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined. Results: Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI‐1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum‐free RPMI‐1640 supported the growth of H. pylori when supplemented with dimethyl‐β‐cyclodextrin (200 μg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD₆₀₀ with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD₆₀₀ contained 1.3 ± 0.1 × 10⁹ CFU/mL. γ‐Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin‐layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours. Conclusions: Thin‐layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.     

The use of a conventional tissue culture plate as an optically accessible perfusion chamber for in situ assays and for long-term cultivation of mammalian cells

An alternative culture system has been developed based on a conventional tissue culture plate (3.5 cm diameter) which is changed into a closed perfusion chamber. The system can easily be scaled up from one to several chambers. The shape and the size of the area of cell growth may be designed to individual experimental demands. The whole culture chamber is optically accessible, so cell growth and morphology can be evaluated by light microscopy. Furthermore the cellular physiology can be characterised by any fluorimetric assay using a bottom type fluorescence reader. A peristaltic pump sustains a constant medium flow through the chamber thus creating true homeostasis. The use of HPLC-valves and connectors allows the switching between different media or assay solutions. Thus it is possible to perform in situ assays also measuring transient effects. A protocol for vitality tests using calcein-AM is worked out for an adherent cell line and for a suspension cell line. The lower detection limits are 7 × 10² cells cm^-2 for the adherent cells and 5 × 10⁴ cells mL^-1 for the suspension cells. The upper limits are 1–2 × 10⁵ cells cm^-2 respectively 8 × 10⁶ cells mL^-1.     

Microorganisms immobilized by membrane inclusion: kinetic measurements in a fixed bed bioreactor and oxygen consumption calculations

Cells living in the pores of macroporous carriers can be immobilized by coating the carriers with a porous membrane. To evaluate the performance of cells immobilized with such a technique, a fixed bed bioreactor was used to study the oxidation of D-sorbitol to L-sorbose by Acetobacter suboxydans. Comparisons were made of immobilized cells to cells living in the pores of a non-coated carrier and to cells living in the absence of a carrier (“submerged cells”). Productivity was similar in all three cultures (4.6–6.3?g sorbose?l^?1?h^?1). Biomass concentration at the outlet was highest for submerged cells (1.3?·?10⁹?cells?ml^?1) but was equal for coated and non-coated carriers (0.4?·?10⁹?cells?ml^?1). Examination of the coated carriers under the electron microscope revealed that only a thin layer near the surface was actually colonized by bacteria. Interestingly, when normalized on the basis of volume, sorbitol oxidation in the colonized layer appeared to be about 100-fold faster than in the bulk medium. A model was derived for oxygen relations inside the coated carriers. This model implicates that the inner parts of the carrier are not colonized by bacteria due to oxygen limitation. The findings indicate that coated carriers have potential to catalyze biotransformations at very high rates, and identify oxygen supply and confinement of cells to the carriers as issues that need further attention. The mathematical model for oxygen concentration profiles inside the coated carriers will be useful for designing improved carriers.     

Critical roles of the TGF-β type I receptor ALK5 in perichondrial formation and function, cartilage integrity, and osteoblast differentiation during growth plate development

TGF-β has been implicated in the proliferation and differentiation of chondrocytes and osteoblasts. However, the in vivo function of TGF-β in skeletal development is unclear. In this study, we investigated the role of TGF-β signaling in growth plate development by creating mice with a conditional knockout of the TGF-β type I receptor ALK5 (ALK5^CKO) in skeletal progenitor cells using Dermo1-Cre mice. ALK5^CKO mice had short and wide long bones, reduced bone collars, and trabecular bones. In ALK5^CKO growth plates, chondrocytes proliferated and differentiated, but ectopic cartilaginous tissues protruded into the perichondrium. In normal growth plates, ALK5 protein was strongly expressed in perichondrial progenitor cells for osteoblasts, and in a thin chondrocyte layer located adjacent to the perichondrium in the peripheral cartilage. ALK5^CKO growth plates had an abnormally thin perichondrial cell layer and reduced proliferation and differentiation of osteoblasts. These defects in the perichondrium likely caused the short bones and ectopic cartilaginous protrusions. Using tamoxifen-inducible Cre-ER™-mediated ALK5-deficient primary calvarial cell cultures, we found that TGF-β signaling promoted osteoprogenitor proliferation, early differentiation, and commitment to the osteoblastic lineage through the selective MAPKs and Smad2/3 pathways. These results demonstrate the important roles of TGF-β signaling in perichondrium formation and differentiation, as well as in growth plate integrity during skeletal development.     

Thin Film Encapsulation of Organic Solar Cells by Direct Deposition of Polysilazanes from Solution

Organic electronic devices (OEDs), e.g., organic solar cells, degrade quickly in the presence of ambient gases, such as water vapor and oxygen. Thus, in order to extend the lifetime of flexible OEDs, they have to be protected by encapsulation. A solution‐based encapsulation method is developed, which allows the direct deposition of the diffusion barrier on top of OEDs, thus avoiding lamination of barrier films. The method is based on the deposition of a perhydropolysilazane (PHPS) ink and its subsequent conversion into a silica layer by deep UV irradiation. The resulting barrier films show water vapor transmission rates (WVTRs) of <10^?2 g m^?2 d^?1 (40 °C/85% relative humidity (RH)) and oxygen transmission rates (OTRs) of <10^?2 cm³ m^?2 d^?1 bar^?1 at ambient conditions. Flexibility of the resulting barrier films is improved by coating a barrier stack of several thin PHPS layers alternating with organic polymer interlayers. These stacks show an increase of WVTR values by less than 10% after 3000 bending cycles. Direct coating of the PHPS films on top of organic solar cells enhances the device lifetime in damp heat conditions from a few hours to beyond 300 h.     

The Metabolism of the Herbicide Diphenamid (N-N-dimethyl-2,2-diphenyl-acetamide) in Cell Suspensions of Soybean (Glycine max)

The fate of the herbicide diphenamid was determined in cell suspensions of soybean [Glycine max (L.) Merr. ‘Wilkin’] at different stages of cell growth: early log phase (3 to 7 d), log phase (7 to 14 d), and stationary phase (14 to 18 d). [Carbonyl-¹⁴C]-diphenamid was added to the suspensions as an acetone solution. Neither diphenamid (2 to 3 μM) nor acetone (0.5% v/v) was phytotoxic. The ¹⁴C-labeled products were identified tentatively by thin layer chromatographic comparison with reference compounds. The major metabolic products formed were N-hydroxymethyl-N-methyl-2,2-diphenylacetamide, N-methyl-2,2-diphenylacetamide, 2,2-diphenylacetamide, and two polar metabolites (0.9 to 25% of the applied ¹⁴C activity) that appeared to be glucose conjugates; one an acidic glucoside. All metabolites were found in both the cell extract and the culture medium, except for the acidic glucoside, which was recovered in small amounts only from the cell extracts. These products were the same as those recovered from intact plants. Similar results were obtained from cell suspensions of different ages. The rate of metabolism by log phase cells was slightly less than the rate for either young or old cells. The results indicated that soybean cell suspensions can be used to obtain reliable information on the fate of agricultural chemicals in soybeans.