Cloning, expression and chromosomal mapping of human lysosomal sialidase and characterization of mutations in sialidosis.

Sialidase (neuraminidase, EC 3.2.1.18) catalyses the hydrolysis of terminal sialic acid residues of glyconjugates. Sialidase has been well studied in viruses and bacteria where it destroys the sialic acid-containing receptors at the surface of host cells, and mobilizes bacterial nutrients. In mammals, three types of sialidases, lysosomal, plasma membrane and cytosolic, have been described. For lysosomal sialidase in humans, the primary genetic deficiency results in an autosomal recessive disease, sialidosis, associated with tissue accumulation and urinary excretion of sialylated oligosaccharides and glycolipids. Sialidosis includes two main clinical variants: late-onset, sialidosis type I, characterized by bilateral macular cherry-red spots and myoclonus, and infantile-onset, sialidosis type II, characterized by skeletal dysplasia, mental retardation and hepatosplenomegaly. We report the identification of human lysosomal sialidase cDNA, its cloning, sequencing and expression. Examination of six sialidosis patients revealed three mutations, one frameshift insertion and two missense. We mapped the lysosomal sialidase gene to human chromosome 6 (6p21.3), which is consistent with the previous chromosomal assignment of this gene in proximity to the HLA locus.

Regulation of phosphate transport by second messengers in capillaries of the blood-brain barrier.

Regulation of phosphate uptake by the blood-brain barrier was studied in isolated bovine capillaries. Dibutyryl cAMP, in the presence of 3-isobutylmethylxanthine, resulted in a dose-dependent inhibition of phosphate uptake. Phosphate influx, with or without 3-isobutylmethylxanthine, was not different. Inhibition of phosphate uptake was also observed when capillaries were preincubated with isoproterenol, parathyroid hormone, insulin and acidic or basic fibroblast growth factors. Treatment of capillaries with vasoactive intestinal peptide, prostaglandin E1, angiotensin II, epidermal growth factor and phorbol esters did not affect phosphate transport. Endothelin I increased phosphate uptake by 15%. Preincubation with cholera toxin also resulted in a dose-dependent decrease in phosphate uptake. In addition, pertussis toxin inhibited phosphate transport by 29%, but only in the presence of 3-isobutylmethylxanthine. These results demonstrate that generation of second messengers, following receptor stimulation, can induce physiological effects on capillary phosphate influx and suggest that G proteins may modulate this transport.

Purification and some properties of liver and brain beta-N-acetyl-hexosaminidase S.

beta-N-Acetyl-hexosaminidase S (2-acetamido-2-deoxy-beta-hexoside acetamido-deoxyhexohydrolase, EC 3.2.1.52) was purified from liver and brain of a patient deceased of type O GM2 gangliosidosis (Sandhoff's disease). Brain beta-N-acetyl-hexosaminidase S was further purified by preparative polyacrylamide gel electrophoresis. The pH optimum of the purified liver and brain enzyme was 5.0 and Km values were 0.8--0.9 mM and 0.3--0.4 mM with 4-methylumbelliferyl-beta-D-N-acetylglucosamine and beta-D-N-acetylgalactosaminide derivatives, respectively. beta-N-Acetyl-hexosaminidase S was thermolabile losing most of its activity after 50 min at 50 degrees C. The apparent molecular weights of the purified liver and brain enzymes were 154 000 and 152 000, respectively. Hexosamines activated beta-N-acetyl-hexosaminidase S whereas the isoenzyme A and B were inhibited. The glycoprotein nature of beta-N-acetyl-hexosaminidase S was suggested by its affinity towards Concanavalin A-Sepharose.

Total parenteral nutrition in the newborn: amino acids-energy interrelationships.

As part of an ongoing study on the influence of intravenous glucose and fat on nitrogen metabolism we evaluated the relationship between the source of infused energy and plasma amino acid levels. Thirty-two studies were performed in 16 appropriate-for-gestational-age newborn infants (birth weight, 2150 +/- 115 g; means +/- SEM). In a crossover design each patient received two 6-d periods of isocaloric and isonitrogenous infusions, differing only by the source of calories (high or low fat intakes). For an energy intake of 80 kcal.kg-1.d-1 (335 kJ.kg-1.d-1) there was a significant hypoaminoacidemia (2338 +/- 185 vs 2937 +/- 196 mumol/L, high fat vs low fat) under the high-glucose intake. These data suggest that above an energy intake of 60 kcal.kg-1.d-1 (251 kJ.kg-1.d-1) there is a threshold at which changes in plasma amino acid levels are triggered by variations in the source of infused energy. Careful examination of all variables, including energy sources, is essential when aminograms are compared.

The composition of preterm milk in relation to the degree of prematurity.

Fifty-one samples of 24-h milk collections obtained during the 1st month of lactation from mothers who delivered after gestations of 26 to 31 wk (VPT) contained higher concentrations of nitrogen (297 +/- 11 mg/dl), total fatty acids (4.46 +/- 0.17 g/dl), percentage medium chain fatty acids (10.8 +/- 0.7), and energy (78.3 +/- 2.0 kcal/dl) than either or both those from 32 to 36 wk (PT) and term (T) gestations. PT collections did not differ from T milk with regard to nitrogen (250 +/- 13 versus 259 +/- 13), total fatty acids (3.94 +/- 0.20 versus 3.20 +/- 0.30), percentage medium chain fatty acids (9.1 +/- 0.5 versus 8.1 +/- 0.7) and energy (69.0 +/- 2.7 versus 66.6 +/- 2.4). Although postpartum age (5 to 10 versus 11 to 30 days) did not change the nutrient and energy content of VPT, PT, and T collections, it is only in 11 to 30 day VPT milk that nitrogen and energy content became higher (p less than 0.05) than either or both PT and T milk. We conclude that the differences in macronutrient composition of PT milk are limited to VPT milk and the data from repeated milk collections in the same mother (28 wk) suggest that there is considerable variability in its composition.

Frequencies of chromosomal abnormalities at amniocentesis: over 20 years of cytogenetic analyses in one laboratory.

Prenatal diagnosis of chromosomal disorders has been performed for more than 20 years, mainly for advanced maternal age. Chromosomal abnormality rates derived from second trimester amniocentesis have mainly come from a collection of small-scale studies from North America and Western Europe. Accurate risk estimates for chromosomal abnormalities are important tools for the physician or obstetrician who would need to make referrals to a prenatal genetic center. This paper presents amniocentesis rates of clinically significant cytogenetic abnormalities for various indications, including advanced maternal age, previous chromosomal abnormality, parental structural rearrangement and a family history of aneuploidy as defined in the text. These data come from a Canadian prenatal diagnosis laboratory with more than 20 years experience in second trimester cytogenetic analysis. They show that the overall frequency of chromosomal abnormalities for advanced maternal age (> or = 35 years) is 1.79%. In this group, 21% of all abnormalities are structural rearrangements (including markers) and less than half of all abnormalities are trisomy 21. The advanced maternal age specific risk of aneuploidies at second trimester is 1.24%. Recurrence risk for aneuploidy after a previous one is 1.29%. However, it is much higher (4.84%) for women of > or = 35 years. When a parent's brother, sister, nephew or niece is affected, the risk of occurrence of aneuploidies (0.24%) is not elevated. When there is a balanced translocation in one of the parents, the overall risk is 10.2% for unbalanced translocations and 37.3% for balanced translocations.

Supernumerary chromosome inherited from a maternal balanced translocation leading to pure trisomy 9p.

We describe a child with a supernumerary chromosome defined as der(9)t(9;22) (q12;p11), resulting in trisomy 9p and trisomy 22p. The mother carried the balanced translocation. In G- and C-banding the derivative chromosome 9 appeared to be dicentric and to contain 22q material. Using in situ hybridization we defined the exact breakpoints of the translocation and ruled out the possibility of a centric fission in the mother's chromosomes.

New autosomal recessive form of amelia.

Amelia is a rare, usually sporadic malformation. We report on a family in which three fetuses had amelia of the upper limbs and variable deficiency of the lower limbs. The fetuses also had minor facial anomalies. Recurrence of the condition in sibs of both sexes suggests autosomal recessive inheritance. Recurrent amelia has been documented in only a few families most often associated with a different set of malformations. Possibly, mutations in more than one gene with different modes of transmission can lead to this severe limb deficiency. We speculate that the mutation found in our cases interferes with formation of the apical ectodermal ridge in the upper limbs and results in its premature degeneration in the lower limbs.

De novo dup(X)(q22.1q25) in a girl with an abnormal phenotype.

Duplication of a portion of Xq has been observed in males with abnormalities. In some cases, their mothers or even grandmothers had the same duplication but did not show any phenotypic abnormalities. However, a few cases of females with a de novo Xq duplication do present some abnormalities. We describe a 16-month-old girl with short stature, motor delay with hypotonia, scoliosis, right hemiatrophy, and ptosis of the right eye, with an Xq duplication. The duplicated region is read dir dup(X)(q22.1q25).

Prenatal detection of intestinal obstructions, aneuploidy syndromes, and cystic fibrosis by microvillar enzyme assays (disaccharidases, alkaline phosphatase, and glutamyltransferase) in amniotic fluid.

Microvillar enzymes (disaccharidases, alkaline phosphatase, and gamma-glutamyltransferase) were assayed in amniotic fluid from pregnancies with normal and abnormal fetuses to determine their specificity and reliability for the prenatal detection of intestinal obstructions and cystic fibrosis. All fetuses with imperforate anus, duodenal atresia, jejuno-ileal atresia, multiple intestinal atresia, or other forms of intestinal obstructions, with or without associated ventral wall defect or aneuploidy syndrome, showed diminished microvillar enzyme activities below the normal range of control amniotic fluid samples. The exclusively intestinal hydrolases maltase, sucrase, palatinase, and alkaline phosphatase were the most reliable and sensitive markers to detect intestinal obstructions whereas more widely distributed trehalase and gamma-glutamyltransferase activities were less sensitive. The combination of intestinal disaccharidase maltase, sucrase or palatinase and ALP assays is more accurate for prenatal diagnosis of CF than a combination of intestinal ALP and GGTF assays.

Autosomal dominant polycystic kidney disease in the fetus.

We report on 3 cases with a fetal presentation of autosomal dominant polycystic kidney disease (ADPKD), which illustrate the variable expression of ADPKD during fetal life. Fetus 1 was diagnosed at 20 weeks of gestation by ultrasonography; a molecular prenatal diagnosis was performed at 10 weeks on fetus 2, a sib of fetus 1; and ADPKD was an incidental finding in fetus 3 who was aborted at 16 weeks for anencephaly. All pregnancies were terminated and pathologic studies of the fetal kidneys were performed. From these cases and a review of the literature, we draw the following conclusions: (1) so far, all fetal ADPKD kidneys that have been histologically studied have shown cystic dilatations; 28/32 of these fetuses had ultrasonographic manifestations of the disease and/or had sibs with an early-onset form of it; (2) these cysts can be found in newly formed nephrons (fetus 2), predominantly in the more mature nephrons of the deep cortex (fetus 1) or more sparsely distributed in the cortex (fetus 3); these different patterns may reflect different rates of progression of the disease; (3) in contrast to the histologic findings in adult kidneys, glomeruli seem to be predominantly affected in fetal ADPKD; (4) severe fetal expression of ADPKD seems to cluster in some families; and (5) so far, all DNA analyses performed in families with subjects presenting during the fetal or neonatal period have been consistent with linkage to the PKD1 locus.

Neuraminidase in cultured fibroblasts and leucocytes of homozygotes and heterozygotes for the mucolipidosis II gene (I-cell disease).

The significance of neuraminidase deficiency reported to be the primary defect in mucolipidosis II has been evaluated by determination of this enzyme activity in cultured fibroblasts, culture medium, and leucocytes from homozygote and heterozygous carriers of the disease. A new and sensitive fluorometric assay of neuraminidase was used with sodium (4-methylumbeliferyl-alpha-D-N-acetylneuraminate) as substrate. We report: 1) nearly total deficiency of neuraminidase in mucolipidosis fibroblasts, 2) partial deficiency of this enzyme in leucocytes of one patient, 3) this decreased activity ceases to exist following Triton X-100 treatment, and 4) intermediary mean neuraminidase activity in fibroblasts and leucocytes from obligate heterozygotes. Although these results would be consistent with the suggestion that neuraminidase deficiency is the primary defect in this disease, evidence from the work of other authors suggests that the enzyme deficiency results from a secondary effect of the mucolipidosis II mutation.

Monozygotic twins with 45,X/46,XY mosaicism discordant for phenotypic sex.

We report on two sets of monozygotic twins (MZTs) discordant for phenotypic sex ascertained at birth when the female twin was noted to have signs of the Ullrich-Turner syndrome. Cytogenetic investigations on the female of the first pair showed 45,X/46,XY mosaicism in lymphocytes but fibroblasts grown from two skin biopsies at separate sites and from gonadal tissue showed only 45,X cells. The male showed mosaicism in both blood lymphocytes and skin fibroblasts. In the second family, both twins also showed mosaicism in lymphocytes. The female had a 45,X karyotype in fibroblasts from skin and gonadal tissue, but in contrast to the first family, the male twin had a normal male karyotype in fibroblasts from skin biopsy and from connective tissue adjacent to the vas deferens. Discordant phenotypic sex in monozygotic twins is rare. As in our cases, the nine previously reported sets of MZTs all had mosaicism for sex chromosome abnormalities. A mitotic error leading to the loss of a Y chromosome prior to, accompanying, or following the twinning process would account for the reported combinations of karyotypes.

Viability assessment by dye exclusion. A fluorescent method for fungal cells.

A new fluorescent staining technique for fungi utilizes the differential affinity of dead cells for rhodamine B. A mounting medium has been devised that includes the dye. This medium allows direct processing of cells from a broth and provides optimal conditions for fluorescence intensity. Comparison with the standard methylene blue exclusion test as applied to the Candida albicans yeast phase indicates similar specificity. Various pseudohyphae and several fungi also exhibited selective uptake of the dye when killed. This technique should prove useful in studying the effect of both drugs and cells on pathogenic yeasts.

Lipoamide dehydrogenase in cultured human skin fibroblasts.

Lipoamide dehydrogenase was identified in cultured skin fibroblasts of normal individuals and patients with Friedreich's ataxia. The optimum conditions for its assay were defined. Data disclosed a normal range of 36--122 mumol/min/mg protein in control fibroblasts and 61--112 mumol/min/mg protein in patients fibroblasts. Numerous precautions should be taken in handling fibroblast cultures for lipoamide dehydrogenase determination.

Fetal intestinal and renal origins of trehalase activity in human amniotic fluid.

Intestinal and renal trehalase isozymes have been distinguished in normal human amniotic fluid on the basis of their membrane-bound character and isoelectric point (pI). The intestinal trehalase was mostly membrane bound in amniotic fluid and had a pI around 4.60. In contrast, the renal form of trehalase was soluble and had a pI around 4.37. These pI values were consistent with those found in extracts of fetal intestinal (pI 4.60) and renal (pI 4.24) tissues. The determination of trehalase isozyme composition of amniotic fluid from pathological pregnancies with anal imperforation and polycystic kidney disease confirmed our findings on the origin of amniotic fluid trehalase. In the sample from a fetus with anal imperforation, low or absent intestinal trehalase isozyme was observed whereas a higher than normal level of renal trehalase activity was found in a fetus with polycystic kidney disease.

N-Acetyl-beta-hexosaminidase isoenzymes of amniotic fluid and maternal serum. Their relevance to prenatal diagnosis of the GM2 gangliosidoses.

The isoenzymes of N-acetyl-beta-hexosaminidase were quantitated in 30 amniotic fluid and 13 maternal serum samples collected between 11 and 40 weeks of gestation using DEAE-Sephadex A-25 chromatography. Isoenzymes A and B consitituted the major components of most amniotic fluids but seven samples characterized by high N-acetyl-beta-hexosaminidase activities contained high proportion of an isoenzyme apparently identical to isoenzyme P of maternal serum. This passage of maternal serum N-acetyl-beta-hexosaminidase into the amniotic cavity could lead to false negative diagnosis of type B and type O GM2 gangliosidoses if the diagnosis rely solely upon isoenzyme analysis in amniotic fluid. However, the release of maternal enzyme into amniotic fluid seems to be restricted to the third trimester of gestation and should not interfere with prenatal diagnosis of the GM2 gangliosidoses ususally performed at an earlier stage of gestation.

Neuraminidase activity in the mucolipidoses (types I, II and III) and the cherry-red spot myoclonus syndrome.

Two neuraminidase (EC 3.2.1.18) comonents, A and B, were distinguished in cultured skin fibroblasts on the basis of thermolability at 37 degrees C. The more labile component (A) t1/2 = 4.7--5.3 min at 37 degrees C, comprises 66--90% of total neuraminidase activity when determined using sodium (4-methylumbelliferyl-alpha-D-N-acetylneuraminate) (MU-alpha-N) as substrate. Activity was assayed at 0 degrees C for 18 h instead of 37 degrees C to fully determine both thermolabile and thermostable components. Diminished activity was noted in cultured fibroblasts from mucolipidoses I, II and III (MLI, MLII, MLIII) and the cherry-red spot myoclonus syndrome (CRSM) patients when assayed at both 0 and 37 degrees C with either MU-alpha-N or each of a series alpha (2 leads to 3)- and alpha (2 leads to 6)-linked N-acetylneuraminyloligosaccharides. Increased sensitivity and rapidity of analyses were achieved using MJ-alpha-N as substrate in determining neuraminidase activity. Results from two obligate heterozygote MLI cell lines (14.5 and 8.0% of control activity) indicate that the MU-alpha-N substrate could be useful for heterozygote detection.

Comparative behavioral, biochemical and pigmentary effects of MPTP, MPP+ and paraquat in Rana pipiens.

We demonstrate that injections of 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP), 1-methyl-4-phenyl-pyridinium ion (MPP+) and Paraquat (PQ+) produce in Rana Pipiens different behavioral, biochemical and skin pigmentation changes. MPTP causes in frogs the main symptoms of Parkinsonism (rigidity, akinesia and tremor) and it darkens the skin of animals. It also decreases brain and, less so, adrenal medulla dopamine. These effects are blocked by Pargyline. MPP+ causes the same symptoms but more rapidly. In contrast, skin pigmentation is clearly lightened. Brain and particularly adrenal dopamine reserves are nearly abolished. Pargyline increases these effects. Paraquat, in a cumulative fashion, eventually causes the same behavioral changes and a slight increase in pigmentation. It initially produces an increase in brain and adrenal dopamine concentrations, but later a significant dopamine concentration decrease. Pargyline potentiates these long term effects, blocks the dopamine increase, but reverses the PQ+ effect upon melanin, producing the same depigmentation as MPP+ alone.

New amphibian models for the study of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

We report the development of two animal models in amphibians (frogs and salamanders) in whom 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces the behavioral (neurological) and biochemical equivalents of the human disease and, in addition, a measurable modification in at least one form of pigment-bearing cell from the neural crest, the skin melanocyte. We propose that this new approach can become an inexpensive, easily quantifiable model for the study of the effect of MPTP on the central and peripheral nervous systems. We also demonstrate that the toxic effect of MPTP can be completely abolished in vivo by treatment with a monoamine oxidase inhibitor and potentiated by an inhibitor of catechol-O-methyltransferase. MPTP is catabolised by oxidation into toxic metabolites, but 1-methyl-4-phenylpyridinium ion (MPP+), the proposed end-metabolite, is even more toxic than MPTP in this model, possibly through a different mechanism.