Heterozygote detection in Fabry disease utilizing multiple enzyme activities.

In Fabry disease, as in other X-linked traits, identification of all heterozygotes is difficult. Reduced plasma alpha-galactosidase activities will correctly identify 60-70% of the carriers. The identification rate improves when an alpha/beta-galactosidase activity enzyme ratio is used. We measured alpha-galactosidase activity in reference to several other enzyme activities, beta-galactosidase, beta-hexosaminidase, and alpha-fucosidase in plasma and leukocytes from 22 suspected and 9 obligate carriers from 4 kindreds of Fabry disease patients. Utilizing such ratios or various combinations of ratios in plasma we have correctly identified the carrier state in 91% of heterozygotes. Leukocyte alpha/beta-galactosidase identified one more female than leukocyte alpha-galactosidase activities alone. We recommend the use of such multiple biochemical tests to identify carriers of Fabry disease.

Role of viscoelastic properties of differentiated SH-SY5Y human neuroblastoma cells in cyclic shear stress injury.

Shear stress and strain lead to neurodegeneration in vivo during head injury, glaucoma, and certain repetitive motion disorders. In vitro, shear stress and strain have been shown to lead to cell injury in a number of models using neurons and neuron-like cells. In the present study we examined the relationship between shear stress, strain, and the extent of cell injury in a cyclic shear stress induced model of cell injury using differentiated SH-SY5Y (human neuroblastoma) cells. Shear stress led to cell strain that increased with increasing stress and diminished upon cessation of shear. Strain rate during cyclic application of shear stress increased by over an order of magnitude from the first to all subsequent cycles, suggesting that the cell and/or its polymer network became more elastic upon cyclic shear stress application. To support this conclusion we measured the degree of cytoskeletal polymerization before and after exposure of cells to cyclic shear stress and found that the fraction of polymerized tubulin in the cell relative to total tubulin decreased by a factor of 2 after six cycles of shear stress. The extent of injury, as indicated by the fraction of cells with fragmented DNA, was three times higher for cyclic shear stress than for steady shear stress and may be related to the change in strain rate and/or cytoskeletal reorganization associated with cyclic stress. These findings may aid in understanding the mechanism by which neurons and neuron-like cells respond to cyclic shear stress and strain and lead to new treatments for disease or injury arising from the exposure of neurons to abnormal cyclic or repetitive stress and strain.

Urinary acidic hydrolases in renal diseases in children.

Acidic hydrolases were assayed in urines of 19 normal children, 33 children with idiopathic nephrotic syndrome of childhood (INS), 21 children with glomerulonephritides (GN) and 7 children with persistent proteinuria/hematuria, and in plasma of 10 children each with INS or GN. Both plasma and urinary acidic hydrolases were studied in intermittent orthostatic proteinuria. Cbeta-galactosidase and Cbeta-N-hexosaminidase were done in normals and children with active renal disease. Significantly (P less than 0.01) elevated urinary acidic hydrolases excretion in active renal diseases, both in INS and GN, returned to a normal range with regression of the diseases. Increased postural proteinuria was associated with normal urinary acidic hydrolases. Both beta-galactosidase and beta-N-hexosaminidase excretion was higher than similar mol wt proteins in normals and increased further in active renal diseases. The data suggests that increased urinary acidic hydrolases is related to the activity of the renal disease, and not to urinary WBC, hematuria or proteinuria. The likely source of urinary acidic hydrolases thus appears to be the injured renal parenchyma itself.

Use of a mathematical model to estimate stress and strain during elevated pressure induced lamina cribrosa deformation.

BACKGROUND: High intraocular pressure (IOP), which is generally associated with glaucoma, causes lamina cribrosa retrodisplacement and deformation. Shear stress and strain resulting from lamina cribrosa deformation have been implicated in tissue remodeling, changes in retinal astrocyte function and retinal ganglion cell (RGC) death observed in vivo during glaucoma. METHODS: A mathematical model was developed to describe the lamina cribrosa exposed to elevated intraocular pressure (IOP). The model is based on the bending theory of plates, incorporates anatomical properties of the lamina cribrosa, and provides estimates of its biomechanical properties. The model relates IOP, the parameter normally correlated with glaucoma, and lamina cribrosa retrodisplacement to stress and strain experienced by cells, parameters that may be more closely associated with cell injury. RESULTS: We estimate that shear strains of 0.05 occur at the edge of a 200 microm thick lamina cribrosa at an IOP of 25 mm Hg. We estimate greater lamina cribrosa deformation and higher shear stress and strain for thinner lamina cribrosa and lamina cribrosa of larger radii. CONCLUSION: These results may provide better estimates of the stress and strain experienced by cells in the lamina cribrosa and may further our understanding of the forces that contribute to optic nerve degeneration during glaucoma.

Pulsatile shear stress leads to DNA fragmentation in human SH-SY5Y neuroblastoma cell line.

1. Using an in vitro model of shear stress-induced cell injury we demonstrate that application of shear to differentiated human SH-SY5Y cells leads to cell death characterized by DNA fragmentation. Controlled shear stress was applied to cells via a modified cone and plate viscometer. 2. We show that pulsatile shear stress leads to DNA fragmentation, as determined via flow cytometry of fluorescein-12-dUTP nick-end labelled cells, in 45 +/- 4 % of cells. No lactate dehydrogenase (LDH) release was observed immediately after injury; however, 24 h after injury significant LDH release was observed. 3. Nitric oxide production by cells subjected to pulsatile shear increased two- to threefold over that in unsheared control cells. 4. Inhibition of protein synthesis, nitric oxide production, Ca2+ entry into cells, and pertussis toxin-sensitive G protein activation attenuated the shear stress-induced cell injury. 5. Our results show for the first time that application of pulsatile shear stress to a neuron-like cell in vitro leads to nitric oxide-dependent cell death.

Aggregation state-dependent binding of beta-amyloid peptide to protein and lipid components of rat cortical homogenates.

Beta-amyloid peptide (A beta) is the primary protein component of senile plaques in Alzheimer's disease. A beta is toxic to neuronal cell cultures, although the mechanism of neurotoxicity is unknown. Neurotoxicity has been correlated to the aggregation state of the peptide. In this work, the synthetic beta-amyloid peptide A beta(1-39) was radioiodinated and fractionated into samples containing varying degrees of aggregated material. Binding of the peptide to rat cortical homogenates (containing both lipids and membrane-associated protein) and to artificial neuronal membrane (containing only lipids) was measured. Binding increased with increasing percent aggregated peptide in the solutions. Aggregated peptide bound to both cortical homogenate and membrane, whereas monomeric peptide bound to homogenate only. These results may help discriminate among alternative mechanisms of neurotoxicity of A beta.

Effect of culture in a rotating wall bioreactor on the physiology of differentiated neuron-like PC12 and SH-SY5Y cells.

A variety of evidence suggests that nervous system function is altered during microgravity, however, assessing changes in neuronal physiology during space flight is a non-trivial task. We have used a rotating wall bioreactor with a high aspect ratio vessel (HARV), which simulates the microgravity environment, to investigate the how the viability, neurite extension, and signaling of differentiated neuron-like cells changes in different culture environments. We show that culture of differentiated PC12 and SH-SY5Y cells in the simulated microgravity HARV bioreactor resulted in high cell viability, moderate neurite extension, and cell aggregation accompanied by NO production. Neurite extension was less than that seen in static cultures, suggesting that less than optimal differentiation occurs in simulated microgravity relative to normal gravity. Cells grown in a mixed vessel under normal gravity (a spinner flask) had low viability, low neurite extension, and high glutamate release. This work demonstrates the feasibility of using a rotating wall bioreactor to explore the effects of simulated microgravity on differentiation and physiology of neuron-like cells.

Effect of beta-amyloid block of the fast-inactivating K+ channel on intracellular Ca2+ and excitability in a modeled neuron.

beta-Amyloid peptide (A beta), one of the primary protein components of senile plaques found in Alzheimer disease, is believed to be toxic to neurons by a mechanism that may involve loss of intracellular calcium regulation. We have previously shown that A beta blocks the fast-inactivating potassium (A) current. In this work, we show, through the use of a mathematical model, that the A beta-mediated block of the A current could result in increased intracellular calcium levels and increased membrane excitability, both of which have been observed in vitro upon acute exposure to A beta. Simulation results are compared with experimental data from the literature; the simulations quantitatively capture the observed concentration dependence of the neuronal response and the level of increase in intracellular calcium.

The role of prion peptide structure and aggregation in toxicity and membrane binding.

Prion diseases are neurodegenerative disorders associated with a conformational change in the normal cellular isoform of the prion protein, PrP(C), to an abnormal scrapie isoform, PrP(SC). Unlike the alpha-helical PrP(C), the protease-resistant core of PrP(SC) is predominantly beta-sheet and possesses a tendency to polymerize into amyloid fibrils. We performed experiments with two synthetic human prion peptides, PrP(106-126) and PrP(127-147), to determine how peptide structure affects neurotoxicity and protein-membrane interactions. Peptide solutions possessing beta-sheet and amyloid structures were neurotoxic to PC12 cells in vitro and bound with measurable affinities to cholesterol-rich phospholipid membranes at ambient conditions, but peptide solutions lacking stable beta-sheet structures and amyloid content were nontoxic and possessed less than one tenth of the binding affinities of the amyloid-containing peptides. Regardless of structure, the peptide binding affinities to cholesterol-depleted membranes were greatly reduced. These results suggest that the beta-sheet and amyloid structures of the prion peptides give rise to their toxicity and membrane binding affinities and that membrane binding affinity, especially in cholesterol-rich environments, may be related to toxicity. Our results may have significance in understanding the role of the fibrillogenic cerebral deposits associated with some of the prion diseases in neurodegeneration and may have implications for other amyloidoses.

The role of G protein activation in the toxicity of amyloidogenic Abeta-(1-40), Abeta-(25-35), and bovine calcitonin.

More than 16 different proteins have been identified as amyloid in clinical diseases; among these, beta-amyloid (Abeta) of Alzheimer's disease is the best characterized. In the present study, we performed experiments with Abeta and calcitonin, another amyloid-forming peptide, to examine the role of G protein activation in amyloid toxicity. We demonstrated that the peptides, when prepared under conditions that promoted beta-sheet and amyloid fibril (or protofibril) formation, increased high affinity GTPase activity, but the nonamyloidogenic peptides had no discernible effects on GTP hydrolysis. These increases in GTPase activity were correlated to toxicity. In addition, G protein inhibitors significantly reduced the toxic effects of the amyloidogenic Abeta and calcitonin peptides. Our results further indicated that the amyloidogenic peptides significantly increased GTPase activity of purified Galpha(o) and Galpha(i) subunits and that the effect was not receptor-mediated. Collectively, these results imply that the amyloidogenic structure, regardless of the actual peptide or protein sequence, may be sufficient to cause toxicity and that toxicity is mediated, at least partially, through G protein activation. Our abilities to manipulate G protein activity may lead to novel treatments for Alzheimer's disease and the other amyloidoses.

Urinary acidic glycohydrolases as an index of kidney damage in juvenile diabetes mellitus.

Two lysosomal glycohydrolases, beta-galactosidase and beta-N-hexosaminidase which have been associated with kidney disease were measured in the urine of 110 youngsters with juvenile diabetes mellitus. The mean enzyme excretions in the diabetic group were intermediate between those of normal youngsters and those with active renal disease. Three youngsters with known kidney disease had elevations comparable to others in the diabetic group but no direct correlation could be shown between enzyme elevations and proteinuria or Addis count abnormalities. Positive correlations were seen between enzyme levels and indices of metabolic balance including blood sugar, cholesterol and triglycerides but not with urine sugar or ketones. Duration and estimated stage and control of diabetes also correlated with the urinary enzymes. These preliminary studies are consistent with the possibility that the excretion of these enzymes reflects the ongoing renal damage which occurs in most juvenile diabetics.

Plasma acidic glycohydrolases in insulin-dependent diabetes mellitus.

We assayed plasma activities of beta-galactosidase, beta-hexosaminidase, alpha-fucosidase and alpha-galactosidase involved in degradation of the glycoprotein molecule in 110 insulin-dependent diabetics aged 3-1/2 to 19 years and compared them to a group of normal youngsters. We correlated the plasma enzyme activities with the duration, control and sequelae of insulin-dependent diabetes. Insulin-dependent diabetics had a significantly higher plasma activity of beta-hexosaminidase and alpha-mannosidase (p less than 0.01) and a significantly lower plasma activity of alpha-fucosidase and alpha-galactosidase (p less than 0.01). Of the 5 enzymes studied, only plasma beta-hexosaminidase correlated with fasting and postprandial blood sugar (p less than 0.01), cholesterol and triglycerides (p less than 0.05). Additionally, poor control of diabetes was also associated with a significantly higher plasma beta-hexosaminidase activity (p less than 0.01). Proteinuria or an abnormal Addis count suggestive of renal involvement was associated with various changes in plasma acidic hydrolases. These changes may be related to insulin deficiency rather than hyperglycemia and may be genetically determined.

Beta-amyloid peptide blocks the fast-inactivating K+ current in rat hippocampal neurons.

Deposition of beta-amyloid peptide (A beta) in senile plaques is a hallmark of Alzheimer disease neuropathology. Chronic exposure of neuronal cultures to synthetic A beta is directly toxic, or enhances neuronal susceptibility to excitotoxins. Exposure to A beta may cause a loss of cellular calcium homeostasis, but the mechanism by which this occurs is uncertain. In this work, the acute response of rat hippocampal neurons to applications of synthetic A beta was measured using whole-cell voltage-clamp techniques. Pulse application of A beta caused a reversible voltage-dependent decrease in membrane conductance. A beta selectively blocked the voltage-gated fast-inactivating K+ current, with an estimated KI < 10 microM. A beta also blocked the delayed rectifying current, but only at the highest concentration tested. The response was independent of aggregation state or peptide length. The dynamic response of the fast-inactivating current to a voltage jump was consistent with a model whereby A beta binds reversibly to closed channels and prevents their opening. Blockage of fast-inactivating K+ channels by A beta could lead to prolonged cell depolarization, thereby increasing Ca2+ influx.

Reduction in cholesterol and sialic acid content protects cells from the toxic effects of beta-amyloid peptides.

beta-Amyloid (Abeta) is the primary protein component of senile plaques associated with Alzheimer's disease and has been implicated in the neurotoxicity associated with the disease. A variety of evidence points to the importance of Abeta-membrane interactions in the mechanism of Abeta neurotoxicity and indicates that cholesterol and gangliosides are particularly important for Abeta aggregation and binding to membranes. We investigated the effects of cholesterol and sialic acid depletion on Abeta-induced GTPase activity in cells, a step implicated in the mechanism of Abeta toxicity, and Abeta-induced cell toxicity. Cholesterol reduction and depletion of membrane-associated sialic acid residues both significantly reduced the Abeta-induced GTPase activity. In addition, cholesterol and membrane-associated sialic acid residue depletion or inhibition of cholesterol and ganglioside synthesis protected PC12 cells from Abeta-induced toxicity. These results indicate the importance of Abeta-membrane interactions in the mechanism of Abeta toxicity. In addition, these results suggest that control of cellular cholesterol and/or ganglioside content may prove useful in the prevention or treatment of Alzheimer's disease.

Fabry's disease in a black kindred.

In a 16-member black kindred with Fabry's disease, four hemizygous males had plasma alpha-galactosidase levels less than 6% and seven heterozygous females had plasma alpha-galactosidase levels between 10% and 50% of normal. A 16-year-old index male had hypertension with left ventricular hypertrophy, abnormal renal function, tortuous retinal veins, "myelin" inclusions in bone marrow macrophages, and intraepithelial inclusion bodies in the kidney. Scrotal angiectasia developed a year after diagnosis. The three other affected males had left ventricular hypertrophy and retinal vein tortuosity. Of the seven carrier females, five had frequent headaches, four had retinal vessel changes, three had proteinuria with normal renal function, and two had bundle-branch blocks on ECGs. There was no deuteranomalopia in this family, although the inheritance pattern of the Fabry gene is X-linked recessive.

Polydipsia, polyuria, and hypertension associated with renin-secreting Wilms tumor.

A 16-month-old black male infant had unusual thirst, polyuria, hyponatremia, and hypertension. His polyuria was unresponsive to vasopressin therapy, and his high blood pressure was not effectively controlled by antihypertensive drugs. Radiographic examinations revealed an occult Wilms tumor in the right kidney. After removal of the tumor, the signs and symptoms were relieved. The tumor had a renin activity about 280 times that of the adjacent renal cortex, and many intracytoplasmic secretory granules were found on electron microscopy. The pathogenesis of these clinical manifestations appears to be mediated through the physiologic pathways of renin-angiotensin II and renin-aldosterone.