The human glucocerebrosidase gene has two functional ATG initiator codons.

Gaucher disease is due to a deficiency in the activity of the enzyme glucocerebrosidase. Glucocerebrosidase is a lysosomal enzyme that presumably requires a signal peptide for transport across the membrane of the rough endoplasmic reticulum and glycosylation for transport into lysosomes. Human glucocerebrosidase cDNA contains two potential ATG start codons in its long open reading frame. The signal peptides that are initiated from each ATG are quite different in their hydrophobicity. We demonstrate that either ATG can function independently to produce active glucocerebrosidase enzyme in cultured fibroblasts. The glucocerebrosidase activity produced from translation products initiated at either ATG is found predominantly in the lysosomes.     

Incidental finding of double minutes (DM), single minutes (SM), homogenously staining regions (HSR), premature chromosome condensation (PCC), and premature centromere division (PCD)?

The incidental finding of DM's, minutes, HSR's, PCC, and PCD in two completely unrelated cases--one is a prenatal diagnosis in a twin pregnancy complicated by hydramnios and feto-fetal exsanguination, the other is an adult Klinefelter patient--raises the question whether such findings are coincidental or whether there is a common denominator in such cases. Possible relationships between these phenomena and the observed cases are discussed.     

Interaction of plasma proteins with negatively charged sites on the pulmonary capillary endothelium of the rat

Summary The endothelial glycocalyx, a polyanionic structure which may regulate the passage of solutes and water through the endothelium, readily binds cationic ferritin (CF). In normal, nonexchange-transfused rats, however, only 7.5% and 6.0% of the luminal plasma membrane and 7.5% and 5.0% of vesicle diaphragms on the thick and thin side of pulmonary capillaries, respectively, bound cationic ferritin. With the graded removal of circulating proteins by exchange transfusion with fluorocarbon emulsion, up to 89 and 82% of the luminal surface, and 76 and 73% of vesicle diaphragms on the thick and thin sides, respectively, bound CF. Although the extent of binding on the thin side was consistently less than on the thick side, the difference was not statistically significant. The extensive binding of CF to the glycocalyx in totally exchange-transfused rats was completely reversible upon addition of lyophilized rat serum protein to the perfusate. These data suggest that in vivo anionic sites of the endothelial glycocalyx are partially masked by adsorbed plasma proteins.     

Crystallization of trimeric recombinant human tumor necrosis factor (cachectin)

Crystals of tumor necrosis factor (TNF) have been obtained in two forms. Rhombohedral crystals grow in 1.8 to 2.0 M ammonium sulfite, pH 7.8 at 21 degrees C, and tetragonal crystals grow in 2.6 M magnesium sulfate, pH 5.5 at 25 degrees C. Analysis of TNF by isoelectric focusing under native and denaturing conditions indicates that TNF molecules exist as trimers in solution. The rhombohedral cachectin crystals belong to space group R3 and have unit cell constants a = b = c = 47.65 A and alpha = beta = gamma = 88.1 degrees. Density determinations and the space group indicate that the unit cell contains one 51,000-dalton trimer. These crystals are stable in the x-ray beam and diffract to at least 1.85 A but are apparently twinned by merohedry. The tetragonal crystals are space group P4(3)2(1)2 or its enantiomorph P4(1)2(1)2 and have unit cell constants a = b = 95.08, c = 117.49. The asymmetric unit contains one trimer; the crystals are stable in the x-ray beam and diffract to beyond 3 A.     

Magnesium and Manganese Content of Halophilic Bacteria

Magnesium and manganese contents were measured by atomic absorption spectrophotometry in bacteria of several halophilic levels, in Vibrio costicola, a moderately halophilic eubacterium growing in 1 M NaCl, Halobacterium volcanii, a halophilic archaebacterium growing in 2.5 M NaCl, Halobacterium cutirubrum, an extremely halophilic archaebacterium growing in 4 M NaCl, and Escherichia coli, a nonhalophilic eubacterium growing in 0.17 M NaCl. Magnesium and manganese contents varied with the growth phase, being maximal at the early log phase. Magnesium and manganese molalities in cell water were shown to increase with the halophilic character of the logarithmically growing bacteria, from 30 mmol of Mg per kg of cell water and 0.37 mmol of Mn per kg of cell water for E. coli to 102 mmol of Mg per kg of cell water and 1.6 mmol of Mn per kg of cell water for H. cutirubrum. The intracellular concentrations of manganese were determined independently by a radioactive tracer technique in V. costicola and H. volcanii. The values obtained by ⁵⁴Mn loading represented about 70% of the values obtained by atomic absorption. The increase of magnesium and manganese contents associated with the halophilic character of the bacteria suggests that manganese and magnesium play a role in haloadaptation.     

THE INFLUENCE OF INTRAVASCULAR FLUID VOLUME ON THE PERMEABILITY OF NEWBORN AND ADULT MOUSE LUNGS TO ULTRASTRUCTURAL PROTEIN TRACERS

The permeability of the alveolar-capillary membrane of newborn and adult mice to horseradish peroxidase (HRP) and catalase was studied by means of ultrastructural cytochemistry, and the permeability to ferritin was studied by electron microscopy. The influence of varying volumes of intravenously injected fluid on the rate of leakage of the tracers from pulmonary capillaries was examined. The tracers were injected intravenously and the mice were sacrificed at timed intervals. Experiments on newborn mice with intranasally instilled HRP were also done. The tissues were fixed in formaldehyde-glutaraldehyde fixative. Chopped sections were incubated in Graham and Karnovsky's medium for peroxidase and in a modification of this medium for catalase. Tissues were postfixed in OsO₄ and processed for electron microscopy. In both newborn and adult mice, the ready passage of peroxidase through endothelial clefts was dependent on the injection of the tracer in large volumes of saline. When the tracer was injected in small volumes of saline, its passage through endothelial clefts was greatly reduced. Endothelial junctions of newborn mice were somewhat more permeable to HRP than those of adult mice. In all animals, alveolar epithelial junctions were impermeable to HRP. Catalase and ferritin did not pass through endothelial junctions. Intranasally instilled HRP in newborn mice was taken up by pinocytotic vesicles and tubules of flat alveolar cells.     

The transport of oxidized glutathione from the erythrocytes of various species in the prescence of chromate

1. Erythrocytes from normal and glucose 6-phosphate dehydrogenase-deficient humans were subjected to hydrogen peroxide diffusion to oxidize the GSH. Studies were carried out in the presence and absence of chromate to inhibit glutathione reductase and with or without the addition of glucose. 2. The GSH content of erythrocytes from other species was oxidized by subjecting them to hydrogen peroxide diffusion in the presence of chromate and glucose. 3. Chromate (1.3mm) inhibited glutathione reductase by about 80%, whereas glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, hexokinase, phosphofructokinase and pyruvate kinase were not inhibited. 4. The GSSG formed was transported from the erythrocytes to the medium. 5. The transport rate of GSSG from glucose 6-phosphate dehydrogenase-deficient erythrocytes subjected to hydrogen peroxide diffusion in the presence of chromate was comparable with that from normal and glucose 6-phosphate dehydrogenase-deficient erythrocytes. 6. The rate of transport of GSSG from erythrocytes of various species studied could be ranked: pigeon>rabbit>rat>donkey>man>dog>horse>sheep>chicken>fish.     

The human glucocerebrosidase gene and pseudogene: structure and evolution

We report the sequence of the entire human gene encoding beta-glucocerebrosidase and that of the associated pseudogene. The gene contains 11 exons extending from base pair 355 to base pair 7232 in the overall sequence. The gene promoter contains TATA- and CAT-like boxes upstream of the major 5' end of the glucocerebrosidase RNA. The two TATA boxes lie between nucleotides (-23)-(-27) and (-33)-(-39) and the two possible CAT boxes reside between nucleotides (-90)-(-94) and (-96)-(-99) in relation to the major 5' end of the mRNA. The functionality of the promoter region was monitored by coupling it to the bacterial gene coding for chloramphenicol acetyltransferase (CAT) and assaying the expression of the enzyme in cells transfected with this vector. The glucocerebrosidase promoter not only directs synthesis of the bacterial enzyme but also exhibits the same pattern of tissue-specific expression as that of the endogenous gene. An apparently tightly linked pseudogene is approximately 96% homologous to the functional gene. However, introns 2, 4, 6, and 7 have large "deletions" consisting of Alu sequences 313, 626, 320, and 277 bp in length, respectively. It is entirely possible that the ancestral gene lacks these sequences and that they have been inserted into the introns of the functioning gene. There is also a 55-bp deletion from a part of exon 9 flanked by a short inverted repeat. The sequence data should facilitate development of methods for diagnosis of Gaucher disease at the molecular level.