The mammary gland, with its hormone-dependent proliferation and development, offers an excellent opportunity to study problems of differentiation. Under hormonal stimulation, this tissue acquires the ability to synthesize specific products characteristic of lactation. Previous workers have demonstrated that insulin added to chemically defined medium maintains the initial histological pattern of midpregnant mammary gland during several days of organ culture. Addition of prolactin to an insulin-hydrocortisone medium elicits an alveolar secretory appearance.1 -Since the histological appearance of the stimulated tissue suggests the alteration of nonsecretory alveolar epithelial cells into highly differentiated secretory cells, we have adapted these organ culture methods to the study of the synthesis of "caseinlike" phosphoproteins by pregnant mouse mammary tissue cultured under various hormonal conditions. Materials and Methods.-Animals: C3H/HeN mice, either virgin or 11-12-day primigravida, were used. After decapitation the abdominal mammary glands were removed bilaterally under sterile conditions and cut into explants ranging in weight from 0.6 to 1.0 mg each. Each experiment was done with tissue from a single animal.Culture methods: For each experiment stock solutions of hormones were made with the following compositions: insulin (Eli Lilly beef zinc insulin, lot 795372), 2.5 mg/ml in 10-3 N HCl; prolactin (NIH-P-S-6, ovine), 2.5 mg/ml in 10-4 N NaOH; hydrocortisone, 8 mg/ml in absolute ethanol. Aliquots from these stock solutions were added to medium 199 (Microbiological Associates) in various combinations to give final concentrations of 5 ug/ml for each hormone. The Pi32 media contained 15 suc/ml, and the C14-amino acid media contained 5 ,uc/ml (Chlorella hydrolysate, specific activity 0.5 mc/mg, obtained from Volk Radiochemical Co.). Sodium penicillin G powder was added directly to the media in most experiments to a final concentration of 35 jug/ml. After all additions, the media were sterilized by filtration through MNillipore filters (0.45 M) into sterile flasks, and stored at 5°if not used immediately.The Fell and Robison culture method as adapted by Chen6 and as previously describedl-3 was used. Two ml of media were added to a watch glass enclosed in a Petri dish. Siliconized lens paper was floated on the media and 3-6 explants were placed on each lens paper (zero time).The Petri dishes were placed in plastic boxes and exposed to 95% air-5% CO2 sufficient to maintain the pH at about 7.4, after which the systems were closed and incubated -at 37°.Nonisotopic media were replaced with isotopic media 4 hr prior to assaying the tissue for "casein." Initial rates of casein synthesis were obtained by placing explants on isotopic media for the first 4 hr of the incubation. Thus, each time point represents the incorporation of isotope during a 4-hr pulse. For systems cultured longer than 2 days, media were replaced with fresh media at 48 hr. D)uplicate culture dishes were used for each determination.Casein assay: At ...

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The structure and biosynthesis of the cell wall of Stapkylococcus aureus, strain Copenhagen, have been investigated previously (cf. reference 1). Studies of the immunological specificity of this strain were undertaken for several reasons. The chemical nature of the surface structure (the cell wall) of this organism was under investigation and had been partially elucidated. If, as seemed likely, the ceil wall contained immunological determinants, the chemical nature of structures responsible for this activity could be defined. Relatively little is known about the chemistry of the antigens ha staphylococci or their relationship to such biological phenomena as immunity, allergy, plmgocytosis, and intracellular lysis. Moreover, it seemed likely that immunological methods would prove useful in further studies of the biosynthesis of the cell wall of S. aureus. These have been the stimuli to the present work.Rabbit anfisera were prepared against whole organisms of S. aureus, strain Copenhagen, and were found to agglutinate cell walls prepared from this species. IIspten inhibition of this agglutination has led to the conclusion that the teichoic acid (eft reference 2) in the cell wall (a polymer of ribitol phosphate which contains glycosidicaUy linked N-acetylglucosamine and esterified D-alanine) contains a serologicaily active group. Among the earliest studies of the antigens in S. aureus strains were those of Julianelle and associates (3, 4). They isolated a substance termed polysaccharide A 25 years ago and showed that it was active both in precipitin tests with rabbit antisera and in inducing wheal and erythema in humans. A similar, but distinct substance, termed polysaccharide B, was isolated from Staphylococcus aIbus strains. In retrospect the described properties, as well as the method of isolation, of polysaccharides A and B indicate that these compounds were teichoic adds. In recent years, immunological activity of the polyglycerophosphate (which belongs to the family of teichoic acids, i.e. polyol phosphate polymers) found in several Gram-positive species has been demonstrated by McCarty (5).