[1] 252w changes in membrane structure in uranyl-treated roots provide such mark- ers. Because uranyl is toxic, our results do not establish pinocytosis as a nor- mal mechanism of intake of material by plants. However, the best evidence of pinocytosis was with exposures to 1 mM uranyl for 1 hour (Fig. 1, F and G) or to 0.1 mM for 20 hours (Fig. I E). Roots were not killed by these treatments; they grew at 30 to 50 per- cent of the normal rate when trans- ferred to water.In animals, salts and proteins induce pinocytosis (8), and this may account for active uptake of soluble macro- molecular substances (9). In plants, a role for pinocytosis in active ion up- take has been postulated (10), and an increase in numbers of pinocytotic vesicles in roots exposed to salts has been reported (11). Chemically characterized plant-root secretions include acidic polysaccharides and protein-polysaccharide complexes of high viscosity (12). Such anionic polymers would not only bind U022+ and other cations but also could account for cell wall swelling and invaginations of the plasma- lemma. Released pinocytotically they could transport bound uranyl to vacu- oles. Freed of uranyl, the carriers could be reused in a cyclic system of uptake. The driving force for such a process could be the creation of complex macromolecules (13). In view of evi- dence that uranyl and calcium ions bind to similar sites (5), our results may represent an exaggerated and abnormal picture of a transport system which functions less conspicuously in normal plants.

[1] 123w tivity. The m The agar or agarose gel pattern of showed three c human fetal LADH consisted of two channel 5) con anodal zones and a single, slow moving to LADH 1, 2, cathodal zone (Fig. 1, channel 1). Fifty fetuses (21 to 260 mm crown-to-rump; estimated gestational age, 45 to 184 -LADH days) showed this pattern (LADH 1) at pH 6.0 to pH 9.0. Later in gestation, a 3 second, less intense, but more cathodal 2 4/ band appeared (LADH 2) (Fig. 1, chan-1 nel 2). This band was also found in is the patterns of most premature infants (870 to 2000 g birth weight). A third, still more cathodal band (LADH 3), varying in intensity, appeared at full 2 3 4

[2] 71w term and was also detected in older Fig. 1. The elec infants (Fig. 1, channel 3). One of the tern of LADH 50 fetal specimens showed, on repeated nates of human study, a single cathodal band with different ages on slightly lower mobility than the normal As much blood fetal band, and this slower band might not contam alcol represent a structural variant of the weighed. A 20 pe "fetal" LADH.

[3] 109w proportion was um Additional evidence that variation enized in 0.25M in banding pattern was related to age 10-'M f-mercaptc came from studies of liver homogenates propyl fluoropho NAD was added I obtained at autopsy from one Caucasian gel. Channel 1,I family consisting of the two parents ture infants; chan and their six children (12). One child infants up to 5-y (aged 3 years) had a pattern similar to 4, 5-to 9-year-ol (aged 3 years).had a pattem shoing year-old children that seen in newborn infants, showing type); channel i LADH bands 1, 2, and 3 (Fig. 1, chantype); channel 7 nel 3). Three of the (aged 5, born infants; cha

[4] 113w ATRUNIVERSITY s) had patterns showing 1 3 as intense bands and ss intense band (LADH the greatest mobility ode (Fig. 1, channel 4). children (aged 11 and tterns like those of their ak LADH bands 1 and LADH 2 band (Fig. 1, cimens from two chilyears) unrelated to the I family had patterns 3-year-old child (Fig. 1, n LADH isozyme pat- d in homogenates from .vers. Both types showed .al zones of LADH ac- iost frequent pattern ,athodal zones (Fig. 1, responding in mobility and 4. In this pattern --GADH - ------_ * . : i ; ŝ w : . Z j e , g : i > % m . 1

[5] 14w trophoretic banding patand GADH in homogelivers and stomachs at agarose gel at pH 8.7.

[6] 68w as possible (blood does ihol dehydrogenase activ- ,ed, and the tissue was brcent (weight to volume) vsed. Tissues were homog-KCI, pH 7.4, containing oethanol and 10-'M diiso- sphate. One milligram of to each 100 ml of agarose fetus; channel 2, premainel 3, full-term, newborn rear-old children; channel Id children channel 5, 11- to adult (most common 6, adult (less common , fetus; channel 8, new- nnel 9, adults.

[7] 66w the LADH 2 band always showed the most intensity, and prolonged electro- phoretic runs suggested that the LADH 1 in some adults moved slightly faster than LADH 1 in fetal specimens. The other frequent adult LADH pattern had only two bands, LADH 2 and 4 (Fig. 1, channel 6). In this pattern LADH 4 was always most intense, and an LADH 1 band was not present.

[8] 77w Homogenates of adult liver specimens obtained by needle biopsy or open sur- gical biopsy showed the same electrophoretic patterns as autopsy specimens from adults. Any given specimen showed the same banding pattern over a 1-year period; and no intercon- version of banding pattern was pro- duced by storage, addition or removal of nicotinamide adenine dinucleotide (NAD) (13), heat, or proteolytic en- zymes. Mixing homogenates of adult and fetal livers produced the sum of the individual banding patterns.

[9] 141w Developmental variation was also noted in the GADH isoenzymes (Fig. 1, channels 7, 8, 9). Homogenates of gastric specimens from embryos (100 to 195 mm crown-to-rump) had a single, low intensity, rapid-moving cath- odal band with a mobility like that of the LADH 4 band in adult liver homog- enates (Fig. 1, channel 7); and therefore this band has been designated GADH 4. In the newborn, a second cathodal GADH band, which moved like LADH 1, appeared and the GADH 4 band increased in intensity (Fig. 1, channel 8). The fetal, newborn, and adult gas- tric isoenzyme patterns were all different from the hepatic pattern at the corre- sponding ages. The adult pattern of GADH consisted of one very rapid and two much slower cathodal bands, the slowest of which was slower than LADH 1 and has been called GADH Is.

[10] 40w No qualitative change in GADH banding pattern was noted after storage for up to 7 months at -20'C. The intensity of GADH bands was strikingly reduced beyond 18 hours postmortem, but again no qualitative change in banding pattern was noted.

[11] 63w Our study, in contrast to an earlier report (6), found no linear correlation between LADH enzyme activity and the crownto-rump length of the fetuses, weight of the newborns, or age of the adults. This difference may be related to the fact that fetal tissue was obtained by -therapeutic abortion in the Scanda- navian study, while spontaneous abortuses comprised the majority of our samples.

[12] 66w The differences in the distribution of LADH enzyme activity in adult, new- born, and fetal samples are statistically significant (X2 = 17.378, .001 <P<.01) (Table 1). The significance is due to dif- ferences in the distribution of adult and newborn samples versus the fetal samples (X2 = 10.827, P<.001). There is no significant difference in the distribu- tion of enzyme levels between adult and newborn samples.

[13] 45w In another investigation, gradient elution from a carboxymethyl cellu- lose column demonstrated three peaks with LADH activity in some individuals and two LADH activity peaks in others (8). This variation was similar to the variation in number of cathodal adult isoenzymes found in our study.

[14] 115w Our results indicate at least three transitional periods in the ontogeny of LADH in man; the first occurs during late fetal life, the second at from 4 to 6 years of age, and the third between 11 and 14 years of age. Although the developmental variation in creatine phosphokinase (4), esterase (3), lactate dehydrogenase (2), and hemoglobins (1) seems to be almost complete or complete by the newborn period, this study indicates that developmental change probably continues for a longer period in human LADH than in other developmental markers thus far de- scribed in man. Since the primary met- abolic function of LADH in man is unknown, no reason can be given for this variation.

[15] 63w Because LADH from human fe- tuses has not-yet been purified, any differences in primary structure be- tween the fetal and adult enzyme are still unknown. However, the finding that, compared with the adult enzyme, fetal LADH has a different Km and optimum pH value as well as a char- acteristic electrophoretic pattern (6) suggests that fetal and adult LADH are structurally different proteins.

[16] 117w Krabbe's globoid cell leukodystrophy is a genetically determined and rapidly fatal neurological disorder of infants, characterized by almost total loss of myelin, severe gliosis, and massive in- filtration of the unique multinucleated globoid cells in white matter. Deficient activity of galactocerebroside /3-galactosidase has been demonstrated in this disease (1). We have extended our study to readily available materials such as peripheral blood and cultured fibroblasts in an attempt to establish a means of antemortem diagnosis of the disease. We also assayed samples from parents of patients with Krabbe's dis- ease, because a partial deficiency of the enzyme would be expected in heterozygous carriers, if the lack of galactocerebroside /3-galactosidase activity is indeed the genetically determined de- fect.

[17] 240w Serum was separated from blood clot by centrifugation, and the enzyme as- says were performed immediately, be- cause both galactocerebroside 83-galactosidase and 4-methylumbelliferyl ,8-galactosidase were unstable in serum and rapidly lost activities even if serum samples were stored frozen or refrigerated (Fig. 1). A similar instability of enzyme in serum was reported for an- other specific galactosidase, galacto- sylgalactosylglucosylceramide: galactosyl hydrolase (2). Leukocyte suspensions were prepared essentially by the method of Snyder and Brady (3), with minor modifications (4). Fibroblasts were cultured from skin (5) and collected by trypsinization. Both leukocytes and fi- broblasts were frozen and thawed sev- eral times before enzyme assays, and fibroblasts were disrupted with high- frequency sound. 83-Galactosidases in leukocytes were stable when kept frozen, with only a slight loss of galactocerebroside /3-galactosidase activity over an 8-week period (Fig. 1). The protein concentrations of leukocyte and fibroblast preparations were determined Abstract. The activity of galactocerebroside /3-galactosidase was extremely low in serum, leukocytes, and cultured fibroblasts of patients with Krabbe's disease. Antemortem diagnosis is possible without organ biopsies. The parents of patients showed enzyme activities generally lower than that of normal controls. This finding provides supportive evidence that the deficient activity of galactocerebroside /8-galactosidase is the genetically determined enzymatic defect underlying the disease. Demonstration of this deficiency requires the use of the specific substrate, galactocerebroside. Assays carried out with synthetic, unnatural substrates, such as 4-methylumbelliferyl /8-galactoside, do not distinguish patients or heterozygous carriers from normal individuals.

[1] 83w or more roots of each of two oat cultivars, Compact (CI 8280) and a Victorgrain mutant (CI 7418), were examined for each treatment, and the illustrations are typical of results with these two varieties. Preliminary work with bean, barley, maize, and wheat roots has yielded results similar to those with oats, but are not extensive enough to justify conclusions about possible differences among species. The procedure for germinating oat seedlings has been published [H. H. Luke and H. Wheeler, Phytopathology 45, 453 (1955)].

[2] 132w 2. The ultrastructure of the cap region of un- treated oat roots has been described ( 14) and is very similar to that of maize and wheat (15). The outer three to five layers of cells of the oat rootcap exhibit high secretory activity and, in this area, dictyosomes are hypersecretory, secretory vesicles are common in the cytoplasm, and intercellular spaces are filled with slime. Occasionally, accumulations of secretory products are found between the cell wall and plasmalemma in secretory cells of untreated oat roots. When treated with uranyl acetate, outer rootcap secretory cells undergo changes quite different from those in cells in the cap interior which normally do not exhibit secretory activity. Because of this, the effects of UO22+ on cells in these two regions will be discussed in separate sections.

[3] 128w The rootcap was chosen for this study partly because its ultrastructural features were well known and partly because it represents a mass of cells isolated from the vascular system of the plant. We emphasize that the course of events found it the rootcap may not apply to other regions of the root. In preliminary work, crystals have been found localized in vacuoles in the root hair region of roots exposed to uranyl for as little as 10 minutes. The fragility of root hairs and their tendency to rupture when placed in hypotonic solutions preclude any conclusion as to how such localization occurred. 3. We wil1 use pinocytosis as a general term for the intake of water or solids by invagination of the plasmalemma as defined by F. A.

[4] 62w Our understanding of the mechanism of differentiation may be advanced by studies which use specific developmental markers such as proteins that are pres- ent in the wild type of a species during a restricted period of development. De- velopmental markers already known in man include embryonic and fetal hemo- globins (1), the lactate dehydrogenases (2), the esterases (3), and creatine phosphokinases (4).

[5] 134w The activity of liver alcohol dehydrogenase (LADH) increases with fetal de- velopment in rats, guinea pigs, and man (5,6). Values of the Michaelis constant (Kms) and optimum values of pH differ between the human fetal and adult forms of this enzyme (6), and develop- mental variation in the isoenzymes of human LADH has also been reported (7). This study shows that the develop- mental variation of human LADH and 0 human alcohol dehydrogenase from gastric mucosa (GADH) continues up to puberty. Tissues were obtained 12 to 36 hours after death. Specimens from spontane- ously aborted fetuses were obtained 12 to 24 hours after passage. An occa- sional fetal specimen was obtained by hysterotomy. All fetuses used had no visible external or internal malformations and tissues were analyzed immedi- ately or stored at -20°to -25°C.

[6] 61w Tissues were homogenized at 0°to 4°C. The homogenate was diluted 1: 5 with buffer prior to enzyme assay (8) and was applied directly to agarose gel for electrophoresis. Agar and agarose gel electrophoresis was performed ac- cording to a modification of the method described by Ursprung (9), and protein concentration was measured by a micro- modification of the Lowry method (10).

[7] 99w Table. 1. Number and proportion of adult, newborn, and fetal samples in different ranges of LADH activity. The mean and range of LADH activity in milliunits per milligram of protein in adults was 79.8 (3 to 319), in newborns was 45.5 (2 to 164), and in fetuses was 15 (1 to 24). Range of LADH Analysis activity in Adult Newborn Fetal milligram of protein No. % No. % No. % 0-14 15 30 7 31.8 12 80 15-79 16 32 1 1 50.0 3 20 80+ 19 38 4 18.2 0 00 x2= 17.378, .001 < P < .o1.

[8] 92w The electrophoresis bands of alcohol de-7, and 81/2 year hydrogenase were identified by com-LADH 1, 2, and parison with partially purified enzyme in addition a les and by comparison of staining intensity 4), which had with and without ethanol. Agar or toward the cath( agarose gel was used to reduce the The two other likelihood that differences in banding 14 years) had pa patterns might be due to the separation parents, with we of different enzyme-coenzyme com-4 and a strong plexes through the sieving properties channel 5). Spe of starch gel (11).

[9] 58w dren (aged 4 y Fifty percent (weight/volume) ho-above-mentioned mogenates from adult human brain, like that of the muscle (skeletal and cardiac), skin, channel 3). spleen, leukocytes, fibroblasts, stomach, Two commor and liver were tested, but only homogterns were foun enates of gastric tissue and liver different adult Iii showed readily defined patterns after two weak anod; the electrophoresis.