Biologic Crystals and Particles Produced in Tissue Culture II
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Biologic Crystals and Particles Produced in Tissue Culture II. Enzymatic Responses and Environmental Transformations* GEORGE G. ROSE (Department of Biology, University of Texas M. D. Ander8on Ho8pital and Tumor In8titute; Department of Medicine (Periodontics), University of Texas Dental Branch; and Tissue Culture Laboratory, Hermann Hospital, Houston, Texas) SUMMARY Unusually large extracellular crystals and particles (BCP) produced by embryo chick cells in special tissue culture environments undergo specific though slow sequences of transformation at 37°C. Observations are recorded by phase-contrast micrographs taken serially over several months. Tubules were found to be the primary observable stage and rhomboids and hexagons the terminal forms. Among the various trans formation pathways two types of intermediate right-handed helices were observed. One type was for rhomboid crystal production and one for hexagonal crystal produc tion. Digestion procedures for six enzymes yielded positive responses to DNase, RNase, trypsin, and pepsin by many of the BCP and negative responses to acid phosphatase and collagenase by all of the BCP. The biologic crystals and particles (BCP) in this report environment. Their slowly changing contours, attitudes, were shown in the first paper of this series (13) as large and sizes were appreciated only when they were photo (up to 300 z in length) RNA-staining objects (pyronin graphed by time-lapse technics which extended over a response; controlled by RNase) which appeared in cul number of days, weeks, and months. The transformation tures of chick embryo tissues viewed by phase-contrast of some types of tubules to right-handed helices was of microscopy. Their pleomorphic shapes were described particular interest. At the time of the first report, it was as either semirigid tubules, right-handed helices (12), not definitely known that the rhomboid and hexagonal ribbons, rhomboid plates, hexagonal plates, or elongated crystalline plates developed from tubules: (a) the rhom (straight or branched) filaments often arranged in whorls. boids were formed by several types of tubular and/or These unusual objects, observed only in a special culture helical transformations and (b) the hexagons developed environment, were closely associated with the embryo from or on the opening ribbons of helices. Many varia chick explants. The special environment was produced tions in the progression of stages have been observed. in multipurpose culture chambers (2, 4—7,13—16)by lay Some of the transformations ifiustrated denote growth and lag broad sheets of dialysis cellophane (8—11,15) across replication processes. Other shapes of the hexagonal the culture-containing coverslip surface. The chamber crystals (triangles, diamonds, and trapezoids) are shown. was thus separated into the following two compartments: Models of the hexagonal system were effected by manipu (a) a shallow explant area (less than 50 js in depth) be lating paper soda straws and are correlated with the specific tween the closely apposing cellophane and culture cover helical morphology observed. slip, and (b) a thick fluid nutrient vault area (3 mm.) between the cellophane and closing coverslip. MATERIALS AND METHODS The purpose of this second report on BCP is to : (a) Embryo chick tis&ues.—Fertile eggs (New Hampshire X identify them with proteins as shown by specific enzy White Leghorns) were supplied by the Texas Agriculture matic digestion and (b) ifiustrate by phase-contrast micro and Mechanical College, Department of Poultry Science. graphs some of their dynamics as observed in the native Upon delivery the eggs were set in a 40°C. incubator; embryos were selected for cultivation after 11—18days of * This work was supported in part by Research Grants (CA 05100-03 CB and CA 06824-01) from The National Cancer Institute incubation. Conventional technics for the sterile dissec of The National Institutes of Health, U. S. Public Health Service; tion of the embryos provided explantable tissues from the grant-in-aid GB-15 Continuation of G-14091 from The National following areas: cerebrum, cerebellum, spinal cord, choroid, Science Foundation administered by C. M. Pomerat of the Pasa thyroid, lung, proventriculus, heart, duodenum, small in dena Foundation for Medical Research; and grant-in-aid from the Easter Seal Research Foundation, National Society for Crippled testine, large intestine, pancreas, kidney, ovary, testis, Children and Adults, Inc. striated muscle, skin, spleen, aorta, pulmonary arteries, Received for publication January 23, 1964. leg bone (periosteum and endosteum removed by scrap 1159 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1964 American Association for Cancer Research. 1160 Cancer Research Vol. 24, August 1964 ing), and frontal bone. Dissected tissues and organs were retention of form in the buffer solution as a control for sub rinsed several times in Earle's balanced salt solution (BSS) sequent enzymatic effects. Following this, the enzyme was and were cut with surgical scalpels into small pieces (ap injected in the following way : a 2-cc. syringe containing ap @ proximately 1 X 1 mm.). proximately ml. of the enzyme mixture was inserted at an Cultivation procedures.—The technic has been ade angle through the rubber gasket so that it was lodged be quately described in earlier reports (2, 4—7,13—16).' tween the cellophane and glass coverslip as it entered the Observation of cultures.—Cultures were observed by culture vault of the chamber. The contents were injected Bausch and Lomb phase-contrast microscopes. Since forcefully into this area, and the needle was withdrawn the chamber coverslip containing the explant is 6—8mm. quickly. A vent needle inserted into the larger vault com above the microscope stage, phase-contrast microscopes pensated for the excess fluid injected. In many cases the with long-working distance condensers were used. Photo cellophane was perforated by such a procedure, and the graphs were made with a Hasseiblad (2@X 2@)still camera BCP immediately flowed through the perforation into the on Agfa Isopan 1FF 13 film and with an EMDECO 16 larger chamber where they were not accessible for satis mm. time-lapse camera unit on Kodachrome hA film. factory evaluation. Repeated efforts permitted successful Fixation procedure for enzyme analyses.—After with results and the entrapment of the enzyme-buffer solution drawal of the nutrient, the chamber was rinsed with BSS between the glass and the cellophane. With extreme care, and refilled with an aqueous solution of KMnO4 (2 per in some cases, the effect of several enzymes could be made cent) for 1 hour. The chamber vault was rinsed with of the same environment. water and opened. Many of the loose particles and crys Miscellaneous tests.—A number of physical, chemical, tals were attached to the coverslip by this type of fixation. and biological tests have been performed on the BCP. It When the cellophane was removed it was apparent that does not appear warranted to detail each of these proced the KMnO4 fixative had variably hard-coated the malle ures, but a summary of results is given in Table 1. able tubules, helices, and ribbons which otherwise were destroyed during removal of the cellophane in unfixed or RESULTS formol-fixed cultures. After water rinsing, the chambers All 23 areas which provided explantable tissues were reassembled without the cellophane but with a few eventually yielded BCP. However, the leg bone cultures, drops of water to prevent drying. Chambers were then generally, were nonproductive. A wide variation in par filled with the various buffers, specific BCP photographed, ticulate number and size occurred among cultures of the and the chamber placed in the incubator for 48 hours to same tissue origin. In this report no attempt will be made ascertain whether the buffer itself produced any effect. to correlate particle size, morphology, or presence in cul In some cases buffers did produce effects on the crystals, tures with specific tissue origin or niass, nutrient factors, but the buffers enumerated below were selected for their PH, or temperature changes. The data principally con inability to disturb the BCP during this interval of time. cern evidence of their protein identity by enzymatic tech Enzymes carried in the appropriate buffer were then ad nics and natural transformations which occurred during mitted into the culture chamber either after the 48-hour extended periods of observation at 37°C. The results of period in the buffer or into new chambers which had not a number of collateral tests are listed in Table 1. had this 48-hour pre-treatment. ENZYMATIC DIGESTION Enzymes and buffers@.— a) Tryp&in: 1—4mg/ml in a phosphate buffer (0.05 M) at The KMnO4 fixation localized the BCP onto the glass pH 7.5 with CaCO3 (0.1 M). coverslips so that they could be photographically followed b) Peps@in: 1—4mg/ml in HC1 (0.02 M) at pH 1.6. periodically during the digestive process, whereas those c) Collagenase: 2 mg/mi in a phosphate buffer (0.05 M) BCP which were unfixed in the environmental compart at pH 7.5. ment between the cellophane and the glass often were d) Acid phosphalase: 2 mg/mi in an acetate buffer (0.05 freely mobile due to the added fluid, and, therefore, difficult M) at pH 5.0. to follow with photography. The responses are sum e) DNase II (acid DNase): 2 mg/mi in an acetate buffer marized in Table 2. (0.008 M) at pH 4.6 with < 0.001 M of Mg@. Trypsin.—Tryptic digestion was notable in members of f) RNase: 2 mg/mi in an acetate buffer (0.05 M)at pH the hexagonal system (Figs. 1—6). The crystals (hexa 6.5. gons) and particles (tubules, helices, and ribbons) Procedure for enzyme analyses of unfixed BCP.—By this responded by a formation of blebs ( 15 minutes to an hour).