An electrophoretic analysis of glycoproteins in the mid-brain fluid of fetal rats

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1 Atlanta University Center W. Woodruff Library, Atlanta University Center ETD Collection for AUC Robert W. Woodruff Library An electrophoretic analysis of glycoproteins in the mid-brain fluid of fetal rats Betty J. Theodore Atlanta University Follow this and additional works at: Part of the Biology Commons Recommended Citation Theodore, Betty J., "An electrophoretic analysis of glycoproteins in the mid-brain fluid of fetal rats" (1979). ETD Collection for AUC Robert W. Woodruff Library. Paper 907. This Thesis is brought to you for free and open access by W. Woodruff Library, Atlanta University Center. It has been accepted for inclusion in ETD Collection for AUC Robert W. Woodruff Library by an authorized administrator of W. Woodruff Library, Atlanta University Center. For more information, please contact

2 AN ELECTROPHORETIC ANALYSIS OF GLYCOPROTEINS IN THE MID-BRAIN FLUID OF FETAL RATS A THESIS SUBMITTED TO THE FACULTY OF ATLANTA UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE '" -r BY BETTY J. THEODORE" DEPARTMENT OF BIOLOGY ATLANTA, GEORGIA MAY 1979 \W

3 Master of Science Thesis of Betty June Theodore Approved: Major Professor > < W\ - Thesis Committee Member Thesis Committee Member Department Chairman / ^ Dean, School of Arts and Sciences

4 ABSTRACT BIOLOGY Theodore, Betty June B.A., Dillard University An Electrophoretic Analysis of Glycoproteins in the Mid-Brain Fluid of Fetal Rats Advisor: Dr. John M. Browne Master of Science degree conferred December, 1978 Thesis dated December 1978 An electrophoretic analysis was carried out on the mid-brain fluid of fetal rats from 13% to 14% days of gestation. This study was conducted to identify and monitor the glycoproteins synthesized during fetalogenesis of the rat brain on days 13% and 14%. In 13% days of gestation the total protein concentration increases from mg/ 100 ml to a mean of mg/100 ml on 14% day of gestation. The densitometric scans of 13% day fetuses mid-brain fluid showed a maximum of 2 bands and 14% day showed a maximum of 3 bands on SDS polyacrylamide gel electrophoresis. Molecular weight determinations of glycoproteins from mid-brain fluid samples of 13% day fetuses yielded readings of 19,600 and 68,000 daltons and 45,000, 26,000, 35,500 daltons from those of 14% day fetuses. The findings of this investigation supports the postulation that glycoprotein concentration and variation in types occur during brain morphogenesis. Consequently these compounds may serve as monitors of brain cell differentiation during rat fetalo genesis. in

5 ACKNOWLEDGEMENTS It is a pleasure to express an infinite amount of gratitude to Dr, John M,, Browne who has so generously rendered his time and services towards assisting me in the accomplishment of this manuscript. Also I would like to thank my mother, Mrs. Betty S. Theodore, family and friends who has given me an endless amount of moral support throughout the duration of this work. IV

6 TABLE OF CONTENTS Page ABSTRACT,,.,,, iii ACKNOWLEDGMENTS,.,..,,.,, XV LIST OF FIGURES,.,,,.,,, vi LIST OF TABLES,, vii Chapter I. INTRODUCTION,,.,.,,..,, t,,,,, 1 II. REVIEW OF LITERATURE 3 III. MATERIALS AND METHODS,.. 5 Sodium Dodecyl Sulfate Polyacrylamide,, 5 Gel Electrophoresis Preparation.,.,,, 5 IV. RESULTS,.,,,,. 8 Total Protein 8 SDS-Gel Electrophoresis Analysis,..,. 8 Molecular Weights Comparisons,.,,.,, 10 V. DISCUSSION 30 VI. SUMMARY AND CONCLUSIONS LITERATURE CITED,.. 35

7 LIST OF FIGURES Figure Page 1. A graph showing changes in total protein of mid-brain fluid from 13% and 14% days of gestation...,,...«: 2. A polyacrylamide gel profile and densitometric scan of rat fetal mid-brain fluid at 13% days of gestation..,..,.,,.<<" < A polyacrylamide gel profile and densitometric scan of fetal rat mid-brain fluid at 13% days of gestation..,.,,..,.,«< < A polyacrylamide gel profile and densitometric scan of fetal rat mid-brain fluid at 13% days of gestation A polyacrylamide gel profile and densitometric scan of fetal rat mid-brain fluid at 14% days of gestation...,..,..., A polycrylamide gel profile and densitometric scan of fetal rat mid-brain fluid at 14% days of gestation A polyacrylamide gel profile and densitometric scan of fetal rat mid-brain fluid at 14% days of gestation...,...,,, A polyacrylamide gel profile and densitometric scan of the protein standard, lactablumin., A polyacrylamide gel profile and densitometric scan of the protein standard, myoglobin,., A polyacrylamide gel profile and densitometric scan of the glycosylated standard N-acetylglucosamine,,..., A polyacrylamide gel profile and densitometric scan of the protein standard, serum albumin., A graph showing a comparison of the molecular weight of two different proteins in the molecular weight range of 17,200. to 68,000. daltons,...!... 29

8 LIST OF TABLES Table 1. Total protein changes in the micubrain fluid of 13% and 14% day old fetal rat,,,,.,,, A electrophoretic profile of the mid^-brain fluid of 13% fetal rat on SDS polyacrylamide gels,., A electrophoretic profile of the mid-brain fluid of 14% day old fetal rat on SDS polyacrylamide gels,.,,',, *,, Summary of the electrophoresis profile of the mid-brain fluid of fetal rats on SDS poly acrylamide gels.,..., A electrophoretic profile of proteins standards on SDS polyacrylamide gels,,,,,,,,,,, Estimated molecular weights of protein fraction of mid-brain fluid from 13% day fetal rats., Estimated molecular weights of protein fraction of mid-brain fluid from 14% day fetal rats VII

9 CHAPTER I INTRODUCTION Glycoproteins characteristically are proteins which have carbohydrates covalently attached as side chains. Carbohydrates which commonly form a part of this chain include N-acetylneuraminic acid (NANA), fucose, galactose, mannose, glucosamine, and galactosamine (Brunngraber, 1969). Thus many possible arrangements of the polysaccharide chains are feasible, which undoubtedly allows for many possible maeromolecular structures. It is because of their hetero geneity (Quarles and Brady, 1971), that glycoproteins are believed to play a role in intercellular recognition during development (Dutton and Barondes, 1970). Glycoproteins are found on the cell surfaces of a wide variety of cell types. They have exclusively been found to exist in nerve endings in mice (Dutton and Barondes, 1970), and rats (Di Benedetta and Cioffi, 1971; Quarles and Brady, 1971). Glycoproteins synthesis is known to be relatively greater in brain tissues of 5-day old rats and 1-10 day old mice. Dutton and Barondes, (1970) and Quarles and Brady, (1971) demonstrated this by injecting radioactive glycoprotein precursors intercerebrally. However glycoprotein ontogeny and transitory composi tion in the mid-brain fluid during development of rat fetuses have not, to our knowledge been identified or characterized. Thus the present studies using polyacrylamide gel electrophoresis in the presence of 1

10 sodium dodecyl sulfate (SDS) has been initiated to identify and monitor the glycoproteins synthesized during the development of the brain of rat fetuses of 13% and 14% days of gestation. An electrophoresis analysis of glycoproteins in the mid-brain fluid of rat fetuses will undoubtedly assist in the elucidation of the role of glycoproteins in brain development. These data, hopefully will further contribute to and/or support and advance current thinking on the role of glycoproteins in brain morphogenesis during rat fetalogenesis. Furthermore the results of these studies will enhance the general knowledge of glycoproteins and their role in intercellular communication in embryonic, regenerating and pathological tissues.

11 CHAPTER II REVIEW OF LITERATURE The composition and formation of the cerebrospinal fluid prior to the early 1930's was thought to have been derived as an ultrafiltrate of the blood. Flexner (1938) comparing the composition of the plasma and the cerebrospinal fluid (CSF) found that the CSF contained higher concentrations of magnesium and chloride ions, and lower concentrations of glucose, proteins, amino acids, uric acid, calcium, phosphate and potassium ions. Flexner concluded that the CSF could not be formed entirely as an ultrafiltrate and that active secretion must be involved. Milhorat (1972), based on carbonic anhydase inhibitors of CSF formation found that approximately 50% of the CSF is formed by secretion and the other half formed as a dialysate of the blood. Weiss (1934) observed secretory activity of the ependymal cells of the embryonic mid-brain of chick embryos on day 8 through 18. He proposed that these ependymal cells were the source of CSF secretion and further suggested that this fluid has an important role in neural tube development. Weed, (1922) postulated that CSF was a product of the ependymal cells of embryonic brain tissues. However, according to Milhorat, (1969) the choroid plexuses contributes to the formation of the CSF. Kapper, (1958) found that during brain morphogenesis the choroid plexuses originates in common with the ependymal epithelium from spongioblasts lining primitive neural tube. Nevertheless present 3

12 evidence as to the exact mechanism of CSF ontogeny and as a consequence the site of CSF formation is still unknown. It is known, however, that a major component of CSF has been found to be glycoproteins. It has been suggested that glycoproteins play a role in the formation of CSF and as a consequence in neural functions. However a precise role for glycoproteins in CSF formation and brain development has not, according to contemporary literature, been eluciated. Recent studies have shown that mammalian brain tissues contain an abundance of sialoglycoproteins especially in the grey matter (Roukema and Heijlman, 1970); they appear to be associated with the membranous elements of synaptosomes; axons and microsomes (Dekirmenjian, et al. 1969; Dekirmenjian and Brunngraber, 1969; Brunngraber, et al. 1917). Brunngraber, (1969) calculated that as much as 5-15% of the total protein content of brain tissue consists of glycoproteins. It has been spectulated (Brunngraber, 1969; Brunngraber, et al., 1967), that the manifold glycoproteins with their great compositional variety of oligosaccharide side chains may play a role in the storage of infor mation or in the transmission capacity of the synaptic region.

13 CHAPTER III MATERIAL AND METHODS Rats fetuses of the Long Evans strain were used throughout this investigation. Female rats in proestrus and estrus were housed with males of the same strain overnight for breeding purposes. The male rat was removed from the cage the following morning and a vaginal smear was taken of the female rat. The appearance of sperm in the vaginal smear was considered as evidence of positive mating and constituted as % day of gestation. Female rats were anaesthetized with ether and sacrificed at 13% and 14% days of pregnancy. The fetuses were removed and the mid-brain fluid was extracted from the diencephalon of each fetus using the technique of Browne, 1970; Browne and Grabowski, The mid-brain fluid of each fetus was pooled and centrifuged in a Beckman 152 microcentrifuged for 5 minutes at 10,000 rpm. The supernatant was separated from the pellet and prepared for electrophoresis. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Preparation For electrophoresis, preparations of 20 ul of the mid-brain fluid was mixed with 10 ul of 0.05% bromophenol blue tracking dye, 1 drop of glycerol, 5 ul of B-mercaptoethanol, and 50 ul of 0.01M sodium phosphate buffer, ph 7.0 containing (0.1%0 SDS and (0.1%) B-mercaptoethanol. The electrophoretic process was conducted in a Gelman 151 Column - Gel Chamber on prepared 10% polyacrylamide gels. Twenty microliters of 5

14 pooled mid-brain fluid containing unknown amounts of protein was layered on top of the gels and electrophoresed at 8ma per gel in a chamber bath containing 0.1% SDS and 0.01M sodium phosphate buffer (ph 7.0) for 2-3 hrs. Prior to staining a needle containing india ink was inserted in each gel at the leading edge of the visible tracking dye. The gels were then rinsed with deionized water and placed in 20% sulfosalicylic acid for 20 to 24 hrs to remove SDS and to fix the proteins. The gels were then placed in 12.5% trichloroacetic acid (TCA) at 65 C for 30 minutes. The gels were subsequently stained with Schiff's Reagent (for glycoproteins) for 30 minutes at 65 C or alternatively stained in Coomassie Brillant Blue (for proteins) for 2-10 hrs at room temperature. Destaining of gels stained with Schiff's is accomplished by two changes of destaining solution (absolute ethanol, Glacial acetic acid, and deionized water), at 65 C for 30 minutes each. The final destaining process is carried out in 10% acetic acid at room temperature. Destain ing of gels stained with coomassie blue was accomplished by placing the gels in a solution consisting of (75 ml of acetic acid, 50 ml of methanol, and 875 ml of deionized water), for a minimum of 15 minutes, but usually requiring 72 hrs. After destaining the gels are stored in 7.5% acetic acid. Once destaining has been accomplished each gel is placed in a small test tube containing storing solution and analysed on a Gelman densitometer to determine the peak profile of each band present. A relative molecular weight of each protein was estimated by plotting the mobility of the sample against the molecular weights of protein

15 standards (Weber and Osborn, 1969). The standard proteins which were used for estimation of molecular weights are given below with the molecular weight value used: lactalbumin, 17,400; myglobin, 17,200; glucosamine, 3,500 and serum albumin, 68,000 daltons. Total protein determinations were conducted using the methods of Lowery, et al. (1951).

16 CHAPTER IV RESULTS Total Protein During fetalogenesis of the Long Evans rats, we have observed that the total protein of the mid-brain fluid shows slight increase from 13% days to 14% days of gestation (Fig. 1,). The total protein was found to be slightly higher on 14% (0,1135 mg/100 ml) compared to ( mg/100 ml) on 13% day (Table 1, Fig, 1), SDS-Gel Electrophoresis Analysis The protein fractions in mid-brain fluid of 13% day sample fluctuated slightly. One fraction was observed in sample A as opposed to two fraction in samples B and C, The relative percent between the fractions of B and C showed variations (Table 2). In sample B, protein fraction number one showed a relative percent of ( gins/ 100 ml) and fraction number two shoed a relative percent of ( gms/100 ml). In sample C, protein fraction number one showed a calculated relative percent of 71,35 (0, gms/100 ml) and in fraction two a relative percent of (0, gms/100 ml). Sample A had one protein fraction which showed a relative protein concentration of gms/100 ml. Figures 2, 3 and 4 shows a polyacrylamide gel profile and a typical densitometeric scans of mid-brain fluid on 13% days of gestation. The protein fraction of three 14% day mid-brain fluid sampl-s also showed some slight fluctuations in banding patterns. Sample A and B had two fraction each and samples C showed three fractions. The relative percent between each fraction also differed (Table 3), In 8

17 sample A protein fraction number one the relative percent was ( gms/100 ml); in fraction number two the relative percent was ( gms/100 ml). In sample B protein fraction number one showed a relative percent of ( gms/100 ml) and fraction number two gave a relative percent of 4.46 ( gms/100 ml). Protein fraction number one of sample C showed a relative percent of 9.48 ( gms/100 ml), protein fraction number two a relative percent of ( gms/100 ml) and fraction number three a relative percent of ( gms/100 ml). Polyacrylamide gel profile and densitometric scans of the mid-brain fluid of 14% days of gestation are in Figs. 5, 6 and 7. Table 4 illustrates a summary of the electrophoretic profile of the mid-brain fluid of fetal rats on SDS polyacrylamide gels.. An electrophoretic profile was also performed on each of the protein standards used (Table 5). Myoglobin and glucosamine showed one protein fraction each. Lactalbumin exhibited two protein fraction. Fraction number one had a relative percent of and fraction number two a relative percent of The profile of serum protein showed three fractions. Protein fraction number one showed a relative percent of 6.06; fraction number two a relative percent of and fraction number three relative percent of Figures 8, 9, 10 and 11 illustrates the polyacylamide gel profile and densitometric scans of these standards.

18 10 Molecular Weights Comparisons The molecular weights, values calculated from SDS polyacrylamide electrophoresis analysis using 10% gels for mid-brain fluid samples from 13% and 14% day old rat fetuses are showed in Table 6 and 7, and figure 12, respectively, The molecular weight of the protein standards are also given (Table 5) and which shows a range from 3,500 to 68,000 daltons. The majority of the suspected glycoproteins from the protein fraction of 13% day mid^brain fluid exhibits an apparent molecular weight of 68,000 daltons for fraction number two, sample B and 19,600 daltons for fraction number two, sample C. The molecular weights for the remaining 13% and 14% day samples were out of the range of the protein standards used, thus they were not reported. The samples of 14% day mid-brain fluid exhibits apparent molecular weights of 34,500 daltons sample A? fraction two; 25,000 daltons sample B, fraction two; 45,000 daltons sample C, fraction two, as estimated by the electrophoretic procedure,

19 Fig. 1. A graph showing changes in total protein of mid-brain fluid from 13% and 14% days of gestation.

20 E o D Gestation (Days)

21 12 Table 1. Total protein changes in the mid-brain fluid of 13% and 14% day old fetal rat. Day Number of Samples Mean (mg/100ml) S.D. Range 13% %

22 13 Table 2. A electrophoretic profile of the mid-brain fluid of 13% fetal rat on SDS polyacrylamide gels. Sample Fraction Relative percent GMS/100ml A Number B Number Number C Number Number

23 Fig. 2. A polyacrylamide gel profile and densitometric scan o rat fetal mid-brain fluid at 13% days of gestation.

24

25 Fig, 3. A polyacrylamide gel profile and densitometxic scan of fetal rat mid^brain fluid at 13% days of gestation.

26 (11

27 Fig. 4, A polyacrylamide gel profile and densitometric scan of fetal rat mid-brain fluid at 13% days of gestation.

28

29 17 Table 3. A electrophoretic profile of the mid-brain fluid of 14% day old fetal rat on SDS polyacrylamide gels. Sample Fraction Relative Percent GMS/100ml Number Number Number Number Number Number Number

30 Fig, 5, A polyacry1amide gel profile and densitometric scan of fetal rat micubrain fluid at 14% days of gestation.

31 00

32 Fig, 6, A polyacrylamide gel profile and densitometric scan of fetal rat mid-brain fluid at 14% days of gestation.

33 cc

34 Fig, 7, A polyacrylamide gel profile and densitometrxc scan of fetal rat mid-brain fluid at 14% days of gestation.

35 C

36 21 Table 4. Summary of the electrophoresxs profile of the mid-brain fluid of fetal rats on SDS polyacrylamide gels. Days of Gestation Sample Fraction Relative Percent GMS/100ml 13% A Number B Number Number C Number Number % A Number Number B Number Number C Number Number Number

37 22 Table 5. A electrophoretic profile on proteins standards on SDS polyacrylamide gels. Sample Fraction Relative Percent Molecular Weight (daltons) Lactalbumin Number ,400 Number Myoglobin Number ,200 N-acetyl- Number ,500 glucosamine Serum Number ,000 Albumin Number Number

38 Fig. 8. A polyacrylamide gel profile and densitometric scan of the protein standard, lactablumin.

39 ( 4

40 Fig. 9. A polyacrylamide gel profile and densitometric scan of the protein standard, myoglobin.

41 t J

42 Fig. 10. A polyacrylamide gel profile and densitometric scan of the glycosylated standard N-acetylglucosamine.

43 k) U

44 Fig. 11. A polyacrylamide gel profile and densitoraetric scan of the protein standard, serum albumin.

45 0

46 27 Table 6. Estimated molecular weights of protein fraction of mid-brain fluid from 13% day fetal rats. Sample Fraction Molecular Weight (daltons) Number 1 Number 1 Number 2 68,000 Number 1 Number 2 19,600

47 28 Table 7. Estimated molecular weight of protein fraction of midbrain fluid from 14% day fetal rats. Sample Fraction Molecular Weight (daltons) Number 1 Number 2 34,500 Number 1 Number 2 25,000 Number 1 Number 2 45,000 Number 3

48 Fig. 12. A graph showing a comparison of the molecular weight of 2 different proteins in the molecular weight range of 17,200 to 68,000 daltons.

49 29.Serum Albumin unknown sample a unknown sample I -^ l unknown sample unknown sample 1 j- unknown sample Myoglobin Mobility

50 CHAPTER V DISCUSSION The ontogeny of cerebrospinal fluid (CSF) has eluded investigators for many years. A survey of the information available shows many incidence of conflicting data resulting in numerous and widely opposed view points. Cerebrospinal fluid was thought by Fremont-Smith (1927) to be an ultrafiltrate of the blood. However Flexner (1938) comparing the composition of the plasma and the CSF concluded that the CSF could not be formed entirely as an ultrafiltrate and that active secretion must be involved. Studnicka (1900) describing secretory activity in the ependymal cells lining the brain ventricles, suggested that the liquefaction around the brain fragments were due to continued activity of their ependymal components. Weiss (1934) stated that the ependymal layers of the chick embryonic brain possess a definite capacity for secreting substances containing proteolytic enzymes and drew a connection between the observed secretion and the production of CSF. Flexner (1938) found that the CSF contained high concentration of magnesium and chloride ions, and low concentrations of glucose, proteins, amino acids, uric acid, calcium, phosphate and potassium ions. Focusing on the protein content of CSF, Bogoch (1959; 1960) prepared from human CSF a non-dialysable glycoprotein. He was able to identify glucosamine, mannose, galactose, fucose and sialic acid. However the actual nature and function of glycoproteins in neural activities is not presently 30

51 31 known. It is known that brain tissue contains high concentration of glycoproteins, Dutton and Barondes, (1970); Di Benedetta and Cioffi, (1971); Quarles and Brady, (1971) have found glycoproteins to exist at neuronal and synaptic surfaces and in subcellular fractions rich in nerve endings in mice and rats, respectively. Also those investi gators have found that glycoprotein synthesis is relatively high in brain tissue of 5 day old rats and 1-10 day old mice. We have observed by electrophoretic studies, total protein analysis and molecular weight determinations that glycoprotein synthesis is occurring in the mid-brain fluid of fetal rats. We referred to cerebrospinal fluid here as mid-brain fluid, since the composition of the fetal rat mid-brain fluid has not been established. Using the Lowry method (Lowry, 1951), we have monitored changes in the total protein of the mid-brain fluid on 13% and 14% days of fetal gestation in Long Evans rats. In the mid-brain fluid of 13% day old fetuses the concentration of the total protein increased from mg/ 100 ml to mg/100 ml on 14% day of gestation. This change in total protein is probably due to metabolic changes which occur normally during embryogenesis. Brunngraber (1969) calculated that as much as 5-15% of the total protein content of brain tissue may consist of glycoproteins. Dutton and Barondes (1969), noted a change in total protein concentration of young mice. Our data showed by electrophoretic profiles of 13% day fetal mid-brain fluid that the glycoproteins are less than in samples from 14% day fetal mid-brain fluid. We suggest that in the mid-brain

52 32 fluid glycoproteins is continously being synthesized, but at different rates during the fetalogenesis. The total protein derived from fetuses at 14*2 days of gestation probably represents not only an increase in glycoprotein synthesis but an increase in its complexity and specificity. According to DiBenedetta and Cioffi (1971), the glycoproteins, in rats 10 days old and older becomes highly complexed and specified. The electrophoretic profile of the mid-brain fluid of three 13% day fetal rats are slightly different. In sample A one band was detected during this period of gestation. In samples B and C two bands were detected. We suggest that the difference between the three 13% day samples exist as a result of one sample being acquired at an earlier or older period in gestation than the other. It is difficult to say because the time of gestation could not be exactly established. Differences in the relative percent from sample to sample may be due to differences in the maternal or in utero environs of each fetus, rendering each sample acquired, physiologically different. This same explanation holds true for the electrophoretic profile of 14% day old fetuses. Interesting though each sample is from a different maternal source, the glycoprotein contents were relatively alike in samples in the same gestational age. Electrophoretic profiles of the protein standards were taken to determine the unknown molecular weights of the suspected glycoproteins of the mid-brain fluid of fetal rats from 13% and 14% days of gestation. On the basis of their mobilities in the SDS polyacrylamide gel, the

53 33 glycoproteins synthesized in developing mid-brain fluid showed molecular weights in the following range. In 13% day fetuses 19,6QQ and 68,000 daltons, and for 14% day fetuses, 34,500, 25,000 and 45,000 daltons. Thus falling in the range of the molecular weights of the standards.

54 CHAPTER VI SUMMARY AND CONCLUSIONS The results of our overall investigation into the electrophoretic analysis of glycoproteins in the mid-brain fluid of fetal rats show that: 1. During fetalogenesis the glycoproteins in the mid-brain fluid of 13% and 14% day fetal rats are constantly flucuating. Glycoproteins in 13% day fetal rat mid-brain are less in content, complexity and specificity than the glycoproteins of 14% day fetal rats. 2. Glycoproteins present in fetal mid-brain fluid may play a major role in cerebrospinal fluid formation and other extracellular functions involved in neurogenesis. 3. The glycoproteins synthesized in the mid-brain fluid of the fetal rats are possibly products of secretory activity since a functional choroid plexus is not definitive during the period when samples were acquired for these studies. 34

55 LITERATURE CITED Bogoch, S. 1969, Glycoproteins of human cerebrospinal fluid. Nature. 184: Bogoch, S Studies on cerebrospinal fluid. J. Biol. Chem. 235: Browne, J. M The effects of embryonic fluids on the morpho genesis of the chick embryo. Ph.D. Dissertation, University of Miami. Browne, J. M. and Grabowski Develop. Biol. (submitted for publication). Brunngraber, E. G Effects of ionic strength of eluting solutions on behavior of sialomucopolysaccharides from rat brain of Sephadex. J. Chromatog. 32: Brunngraber, E. G., Dekirmenjian, H., and Brown, B. D The distribution of protein-bound N-acetylneuraminic acid in subcellular fractions of rat brain. Biochem. J. 103: Brunngraber, E. G The possible role of glycoprotein in neural function. Perspect. Biol. Med. 12: Dekirmenjian, H., Brunngraber, E. G., Lemkey, J. W. and Larramend, L. H Distribution of gangliosides, glycoprotein-nana and acetylcholinesterase in axonal synaptosomal fractions of cat cerebellum. Exp. Brain. Res. 8: Dekirmenjian, H., Brunngraber, E. G Distribution of N- acetylneuraminic acid in subcellular particulate fraction prepared from rat whole brain. Biochem. Biophys. Acta. 177:1-10. DiBenedetta, C. and Cioffi, L. A Glycoproteins during the development of the rat brain. Adv. Exper. Med. and Biol. 25: Dutton, G. R. and Barondes, S. H Glycoprotein metabolism in developing brain. J. Neurochem. 17: Flexner, L. R Changes in the chemistry of the cerebrospinal fluid during fetal life in the pig. Amer. J. Physiol. 124: Fremont-Smith, F Arch. Neurol Psychiat., 17:317, as quoted by Milhorat, T. H Hydrocephalus and cerebrospinal fluid, Williams and Wilkins Co., Baltimore, p

56 36 Kapper, J. A Structure and functional changes in the telecephalic choroid plexus during human ontogenesis. In cibia foundation symposium on the cerebrospinal fluid. (G. E. Wolstenholme and C. M. O'Connor, ed,) Little, Brow and Co.? Boston, p. 3. As quoted by Milhorat, T. H, Hydrocephalus and cerebrospinal fluid, Williams and Wilkin Co., Baltimore, p Lowry, 0. H., Rosebrough, N., Farr, L. and Randall, R. 1951, Protein measurement and folin phenol reagent. J. Biol. Chem. 193: Quarles, R. H. and Brady, R Synthesis of glycoprotein and gangliosides in developing rat brain. J. Neurochem, 18: Studnika, S Untersuchugen u den Bau des Ependyms der nervosen Zentralorgane. Anat. Hefte, Bd. 15, S. 301, as quoted by Weiss, P Secretory activity of the inner layer of the embryonic mid-brain of the chick as revealed by tissue culture. Anat. Rec. 58: Weber, K. and Osborn, M The reliability of molecular weight determination by sodium dodecyl sulfate polyacrylamide gel electrophoresis. J. Biol. Chem. 244: Weed, L. H The cerebrospinal fluid. Physiol. Rev, 2: Weiss, P Secretory activity of the inner layer of the embryonic mid-brain of the chick as revealed by tissue culture, Anat. Rec. 58: