Human fl-glucuronidase (fl-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycoprotein lysosomal hydrolases, is specifically taken up from the culture medium by human fibroblasts. Prior work has indicated that the enzyme exhibits charge heterogeneity and that "highuptake" forms, i.e., those rapidly internalized by human fibroblasts, are more acidic than, slowly internalized forms. Here we present two lines of evidence that the acidic group required for the high-uptake property of certain forms of the enzyme is a phosphate on, or in proximity to, a D-mannose-type carbohydrate. The first line of evidence was obtained from analysis of inhibition of enzyme pinocytosis by yeast mannans, phosphorylated sugars, and sugars. Mannans that contained phosphate were more potent inhibitors than those that did not contain phosphate. iD-Mannose 6-phosphate was a more potent inhibitor than either D-mannose 1-phosphate or 2-deoxy-D-glucose 6-phosphate. D-Mannose and certain related sugars were weak pinocytosis inhibitors, while 2-and 4-epimers of mannose were noninhibitory. Competitive inhibition was demonstrated and the apparent K is estimated for the following compounds: Saccharomyces cerevisiae mannan from mutant X2180-mnnl, 3 X 10-6 M; mannan from wild-type S. cerevisiae, 3 X 10-5 M; D-mannose 6-phosphate, 6 X 10-5 M; L-fucose, 4 X 10-2 M; and D-mannose, 6 X 10-2 M. The second line of evidence comes from the observation that alkaline phosphatase [orthophosphoricmonoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] treatment of human platelet fl-glucuronidase abolished its "high-ptake" activity, without diminishing its catalytic activity, and converted some forms of the heterogeneous enzyme to less acidic forms.Pinocytosis of lysosomal enzymes, identified by Neufeld and coworkers (1) as uptake of corrective factors by enzyme-deficient fibroblasts, displays the selectivity and saturability expected for a receptor-mediated process (2). Several investigators (3-5) have studied kinetic aspects of this process with enzymes from different sources. Hickman and Neufeld (6) suggested that many lysosomal hydrolases have similar components that are essential for their recognition and uptake by human fibroblasts. This suggestion was based on the observation that fibroblasts from patients homozygous for a single gene mutation (I-cell disease) secrete several hydrolases which are not specifically pinocytosed (6). The finding that periodate treatment of fihexosaminidase secreted by normal fibroblasts destroyed its uptake activity led to the suggestion that the "recognition component" on the enzyme contains carbohydrate (7).We have utilized fl-glucuronidase (f3-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) to study this pinocytosis process. Previously published studies have shown (i) that human f,-glucuronidase from all tissue sources investigated exhibits charge heterogeneity (8); (ii) that high-uptake forms, which are specifically pinocytosed by fibroblasts, are more acidic than the low-uptake, or poorly ...

A B S T R A C T In the course of a controlled study to evaluate different forms of immunotherapy for subjects with insect-sting hypersensitivity, we observed 11 subjects who had systemic cutaneous urticarial reactions and 3 subjects who experienced systemic anaphylaxis. With the exception of tachyeardia, there were no cardiopulmonary changes in the subjects with urticaria, whereas the major manifestation of anaphylactic shock in the other three subjects was severe hypotension that was probably secondary to peripheral vasodilation. Significant abnormalities in gas exchange developed in two subjects. In one, bronchospasm precipitated a respiratory arrest followed by endotracheal intubation with mechanical ventilation. Although plasma histamine levels were not related to the development of cutaneous reactions, the plasma histamine levels correlated with the severity and duration of the cardiopulmonary changes observed during anaphylactic shock. The two subjects with the most severe shock showed evidence of intravascular coagulation characterized by a diminution of Factor V, Factor VIII, fibrinogen, and high molecular weight kininogen, as well as changes in components of the complement system. Standard therapy with epinephrine and fluids, usually recommended for the treatment of systemic anaphylaxis, did not immediately reverse either the hemodynamic or the respiratory abnormalities in the two subjects with the most severe

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A B S T R A C T We recently presented data showing that mannose-6-phosphate was a potent competitive inhibitor of pinocytosis of human platelet f8-glucuronidase, and that treatment of "high-uptake" forms of the enzyme with alkaline phosphatase destroyed the high-uptake property of the enzyme without diminishing its catalytic activity. These data indicate that phosphate is a necessary component of the recognition marker on the enzyme for pinocytosis by human fibroblasts, and suggest that the phosphate on highuptake forms of the enzyme is present as a phosphohexosyl moiety. Results presented here show that mannose-6-phosphate is also a potent inhibitor of pinocytosis of the following enzyme preparations: (a)

Although amyloid beta (Abeta) deposition has been a hallmark of Alzheimer's disease (AD), the absence of a phenotype in the beta amyloid precursor protein (APP) knockout mouse, tends to detract our attention away from the physiological functions of APP. Although much attention has been focused on the neurotoxicity of Abeta, many studies suggest the involvement of APP in neuroplasticity. We found that secreted amyloid precursor protein (sAPP) increased the differentiation of human neural stem cells (hNSCs) in vitro, while an antibody-recognizing APP dose-dependently inhibited these activities. With a high dose of sAPP treatment or wild-type APP gene transfection, hNSCs were differentiated into astrocytes rather than neurons. In vivo, hNSCs transplanted into APP-transgenic mouse brain exhibited glial differentiation rather than neural differentiation. Our results suggest that APP regulates neural stem cell biology in the adult brain, and that altered APP metabolism in Down syndrome or AD may have implications for the pathophysiology of these diseases.