Erythroid cell differentiation and the inhibition of cytokinesis by cytochalasin.

Cytochalasin B produces multinucleated erythroid cells in tissue cultures of very young chick blastoderms. There is no apparent qualitative interference with differentiation and maturation of erythroid cells, but the amounts produced are reduced 4- and 10-fold. These effects of cytochalasin are readily reversible.

Characterization of neuraminidase activity of cultured human fibroblasts.

Investigations have been carried out to establish the enzymatic properties and specificities of the neuraminidase of cultured human fibroblasts. Homogenates of these cells cleaved the actylated derivative of neuraminic acid from fetuin, N-acetylneuraminyllactose and 2-(3' methoxyphenyl)-N-acetyl-alpha-neuraminic acid. Maximum activity occurred between pH 4.2 and 4.6 in sodium acetate buffer. The Km values were 3.6 . 10(-4) M, 3.0 . 10(-3) M and 1.1 . 10(-3) M, respectively, against fetuin, N-acetylneuraminyllactose and 2-(3'methoxyphenyl)-N-acetyl-alpha-neuraminic acid. Against the first two substrates, the rate of hydrolysis fell below the expected value as the cell homogenate was diluted with water or 10 mM NaCl. Dilution with 8 mg/ml bovine serum albumin prevented the deviation and yielded the expected linear decrease. After the first 2-h incubation, the rate of hydrolysis decreased from the initial linear rate. The enzyme(s) was partially or completely inactivated by sonication at 20 kHz, freeze-thaw treatment, incubation at 52 degrees C or storage for 48 h at -70 degrees C. Suspension of the fibroblasts in water for 10 min at room temperature, followed by homogenization with a tissue grinder, yielded preparations that were suitable for the assay of the neuraminidase activity.

Accumulation of N-acetylneuraminic acid (sialic acid) in human fibroblasts cultured in the presence of N-acetylmannosamine.

Human skin fibroblasts incubated for 72 h in medium containing 10 mM N-acetyl-D-mannosamine accumulate material that yields a chromophore in the presence of thiobarbituric acid. This material was tentatively identified as free (unbound) sialic acid due to its reactivity with thiobarbituric acid prior to acid hydrolysis, its solubility in 10% trichloroacetic acid, its chromatographic properties on an anion-exchange column and its enzymatic susceptibility to acylneuraminate pyruvate-lyase. Mass spectrometry analysis established that the accumulated material was, in fact, N-acetylneuraminic acid. Loading studies demonstrated a linear relationship between the amount of N-acetylmannosamine in the medium and the level of sialic acid accumulating within the cells. Cells grown in the absence of N-acetylmannosamine contained an average of 5 nmol free sialic acid/mg protein, while cells cultured for 72 h in 20 mM amounts of this material contained an average of 156.3 nmol free sialic acid/mg protein. When the cells were removed from the N-acetylmannosamine-enriched medium and incubated in regular medium, more than 80% of the accumulated, intracellular sialic acid disappeared within the first 96 h. It was concluded from these data that normal fibroblasts cultured in medium enriched with N-acetylmannosamine store large amounts of N-acetylneuraminic acid and can thus serve as an excellent model for the study of both normal and abnormal sialic acid metabolism, transport, storage and/or metabolic (feedback) regulation in human tissue.

Mannosidosis: deficiency of a specific alpha-mannosidase component in cultured fibroblasts.

Evidence is prisented which shows that the enzymatic defect in mannosidosis, the deficiency of alpha-mannosidase, is expressed in cultured skin fibroblasts from patients with mannosidosis. Additionally, by Cellogel electrophoresis, the enzyme can be separated into two major components (a heat stable component missing in mannosidosis fibroblasts with a pH optimum of 3.6-4.0 and a heat labile component present in mannosidosis fibroblasts with a pH optimum of 5.6-6.0). The specific activity and electrophoresis of alpha-mannosidase from cultured amniotic fluid cells are also shown to be similar to cultured skin fibroblasts.

Focal cerebral ischemia during anesthesia with etomidate, isoflurane, or thiopental: a comparison of the extent of cerebral injury.

An investigation was performed to compare the cerebral protective properties of etomidate, isoflurane, and thiopental. In separate groups of spontaneously hypertensive rats, etomidate, isoflurane, or thiopental was administered to achieve and maintain burst-suppression of the electroencephalogram (3-5 bursts/min) for the duration of the experiment. A fourth group received 1.2 minimal alveolar concentration halothane. All groups underwent 3 hours of middle cerebral artery occlusion and then 2 hours of reperfusion. Thereafter, the animals were killed and the volume of injured brain was determined by staining with 2,3,5-triphenyltetrazolium. Physiological parameters did not differ among the four groups during the investigation, with the exception that hemolysis occurred in the etomidate group (free hemoglobin levels, approximately 0.4 g.dl-1). The volume of injured brain in the thiopental group (56 +/- 10 mm3) was significantly smaller than that in the halothane control group (99 +/- 13 mm3). The volumes of injured brain in the etomidate and isoflurane groups (145 +/- 11 mm3 and 139 +/- 14 mm3, respectively) were significantly larger than those in the control and thiopental groups. We speculate that the apparently detrimental effect of etomidate may be the result of the binding of nitric oxide of cerebral endothelial origin by the iron component of free hemoglobin. Intracranial pressure was not recorded, and in the isoflurane group, there may have been adverse effects on cerebral perfusion pressure associated with vasodilation caused by high concentrations of isoflurane. The results are consistent with a protective effect by barbiturates.

Overproduction of N-acetylneuraminic acid (sialic acid) by sialuria fibroblasts.

Fibroblasts from the original sialuria patient are shown to contain high levels of free sialic acid, i.e., an average of 87 versus a normal average of 2 nmol/mg of protein. Gas liquid chromatography-mass spectrometry analysis confirmed that the accumulated material is N-acetylneuraminic acid. Addition of D(+) glucosamine (0-5 mM) to the media of these cells increased the intracellular free sialic acid concentrations from 74 to 137 nmol/mg protein. In contrast, normal cells treated in an identical manner maintained their normal free sialic acid level of less than 4 nmol/mg protein. Addition of 20 mM N-acetylmannosamine in place of glucosamine resulted in a marked increase in free sialic acid in both the patient and the control, i.e., increases of 157 (from 95 to 252) and 120 (from 3 to 123) nmol/mg protein, respectively. Finally, while normal cells in the presence of glucosamine accumulated high levels of uridine diphosphate N-acetylhexosamine, the patient's cells accumulated much lower amounts of this compound. It is concluded that the elevated sialic acid level in sialuria is due, at least in part, to overproduction of free N-acetylneuraminic acid. Indirect evidence is presented that this may result from either hyperactivity or increased levels of the enzyme (uridine diphosphate N-acetylglucosamine 2-epimerase) that has been shown in other tissues to convert uridine diphosphate N-acetylglucosamine to N-acetylmannosamine.

Correction of sphingomyelinase deficiency in Niemann-Pick type C fibroblasts by removal of lipoprotein fraction from culture media.

The average sphingomyelinase activity of fibroblasts obtained from 12 Niemann-Pick type C patients was 37.9% of that of normal fibroblasts (27.2 versus 72 nmol (mg protein)-1 h-1) when the cells were cultured in minimum essential medium containing 13% fetal bovine serum. Following replacement of the above medium with medium in which the lipoprotein fraction had been removed from the fetal bovine serum, the sphingomyelinase activity rose over a 7-day period from about 1/3 of normal to normal or above. Upon reintroduction of medium containing 10% fetal bovine serum which had not been extracted, the sphingomyelinase activity of the Niemann-Pick type C cells again fell within 48 h to 30% of the normal controls. In contrast, cell lines from patients with either Niemann-Pick Type A or B were not influenced by the presence or the absence of lipoprotein, i.e. lacked sphingomyelinase activity under all culture conditions examined. Histochemical staining with filipin showed an inverse relationship between the sphingomyelinase activity and intracellular, free, unesterified, cholesterol level. Moreover, immunochemical staining with an antibody against a lysosomal membrane protein provided direct evidence that the accumulation of unesterified cholesterol in cells cultured in regular (non-extracted) medium occurred within lysosomes and/or related organelles.

Six novel deleterious and three neutral mutations in the gene encoding the alpha-subunit of hexosaminidase A in non-Jewish individuals.

Initial investigations demonstrated that only 3/34 "Tay-Sachs chromosomes" in 22 unrelated, non-Jewish patients or carriers of some form of GM2-gangliosidosis (7 black and 15 non-Jewish Caucasian) had either of the two mutations commonly found in the Jewish population. To determine the nature and incidence of the alterations in this non-Jewish population we have utilized PCR, single-strand conformation polymorphism analysis and sequencing to detect new mutations in genomic DNA. Fourteen primer sets have been utilized to analyze 80% of the coding region and 23/26 splice sites of the gene coding for the alpha chain of hexosaminidase A. Presumed deleterious mutations were discovered in 17/34 chromosomes believed to be carrying a beta-hexosaminidase A alpha-subunit gene mutation. Ten had abnormalities which have been described previously. In the remaining 24 Tay-Sachs disease alleles, six novel mutations predicted to be deleterious were discovered. These include two small deletions (a single-base frameshift and a three-base deletion removing an amino acid), two different nonsense mutations, an initiation codon mutation (ATG----GTG), and a missense mutation (Arg499Cys) in a highly conserved residue. In addition, three presumed nondeleterious mutations were found.

Approaches to the control and prevention of Tay-Sachs disease.

PIP: A prototype Tay-Sachs disease prevention program is presented and di scussed. The program strategy, design, and methodology are reviewed and results and analyses of epidemiologic data, testing results, and in-prog ram evaluations of the testing method are given, along with overall program results. Psychosocial, genetic, and socioeconomic consideration s in the counseling of screenees and relatives of carriers are discussed. In the 1st year of the program, 11 couples, none of whom had previously had a Tay-Sachs child, were identified to be at risk for Tay-Sachs disease in their offspring.^ieng

Enhanced fluoride uptake by teeth through use of iontophoretic techniques.

The effects of electric currents on the uptake of fluoride ions by human teeth, in vitro were determined by measuring the change in fluoride ion concentration of the electrolyte in which teeth were subjected to the electrochemical treatment. There was an enhanced uptake when the gold electrode embedded in the tooth was polarized anodically. Evidence that the fluoride ions were taken upon by the teeth was shown by subsequent cathodic polarization of the gold electrode in a fluoride free electrolyte and by measurement of fluoride release into the solution.

Neuraminidase deficiency in the original patient with the Goldberg syndrome.

Homogenates of cultured skin fibroblasts from a non-ambulatory, 20-year-old male with cherry-red spots, corneal clouding, seizures, mental retardation, dysostosis multiplex, dwarfism, coarse facies and loss of vision, originally described by Goldberg et al. (1971), have diminished neuraminidase activity and an excess of neuraminic acid-rich compounds. Specifically, these cells have 2-17% normal neuraminidase when measured with 2-(3' methoxyphenyl)-N-acetyl-alpha-neuraminic acid, N-acetyl-neuramin-lactose and fetuin. Activities of 12 other lysosomal enzymes were either at or above the range of normal control fibroblasts. Total neuraminic acid concentration was 44.3 nmol/mg protein versus an average control value of 14.2. It is concluded that the Goldberg syndrome should be considered, along with mucolipidosis I and the cherry-red spot -- myoclonus syndrome, as resulting from a primary neuraminidase deficiency.

Segregation within a family of two mutant alleles for hexosaminidase A.

A healthy adult female was found to have low levels of hexosaminidase A in serum, leukocytes and fibroblasts when these were assayed with artificial substrates. Fibroblast assay with GM2 ganglioside gave values consistent with a Tay-Sachs heterozygote. Studies of this non-Jewish family revealed evidence for segregation of two mutant alleles for hexosaminidase A.

Experimental subarachnoid hemorrhage in rats: effect of intravenous alpha-alpha diaspirin crosslinked hemoglobin on hypoperfusion and neuronal death.

BACKGROUND: Hemodilution with diaspirin crosslinked hemoglobin (DCLHb) ameliorates occlusive cerebral ischemia. However, subarachnoid hemoglobin has been implicated as a cause of cerebral hypoperfusion. The effect of intravenous DCLHb on cerebral perfusion and neuronal death after experimental subarachnoid hemorrhage was evaluated. METHODS: Rats (n = 48) were anesthetized with isoflurane and subarachnoid hemorrhage was induced by injecting 0.3 ml of autologous blood into the cistema magna. Each animal received one of the following regimens: Control, no hematocrit manipulation; DCLHb, hematocrit concentration decreased to 30% with DCLHb; or Alb, hematocrit concentration decreased to 30% with human serum albumin. The experiments had two parts, A and B. In part A, after 20 min, cerebral blood flow (CBF) was assessed with 14C-iodoantipyrine autoradiography. In part B, after 96 h, in separate animals, the number of dead neurons was determined in predetermined coronal sections by hematoxylin and eosin staining. RESULTS: Cerebral blood flow was greater for the DCLHb group than for the control group; and CBF was greater for the Alb group than the other two groups (P < 0.05). In one section, CBF was 45.5 +/- 10.9 ml x 100 g(-1) x min(-1) (mean +/- SD) for the control group, 95.3 +/- 16.6 ml x 100 g(-1) x min(-1) for the DCLHb group, and 138.1 +/- 18.7 ml x 100 g(-1) x min(-1) for the Alb group. The number of dead neurons was less in the Alb group (611 +/- 84) than in the control group (1,097 +/- 211), and was less in the DCLHb group (305 +/- 38) than in the other two groups (P < 0.05). CONCLUSIONS: These data support a hypothesis that hemodilution decreases hypoperfusion and neuronal death after subarachnoid hemorrhage. The data do not support the notion that intravascular molecular hemoglobin has an adverse effect on brain injury after subarachnoid hemorrhage.

Sialidase (alpha-n-acetyl neuraminidase) deficiency: the enzyme defect in an adult with macular cherry-red spots and myoclonus without dementia.

A 31-year-old male is described who has macular cherry-red spots, increased deep tendon reflexes and and myoclonus without dementia. An older brother died at age 33 of a disease with similar symptomatology. Homogenates of cultured fibroblasts from the patient exhibited 2.6, 8.1 and 12.4% of normal mean sialidase (neuraminidase, N-acetyl-neuraminosyl glycohydrolase, EC 3.21.18) activity, respectively, against 2-(3'-methoxyphenyl)-N-acetyl-alpha-neuraminic acid, N-acetyl-neuramin-lactose and fetuin. Activities of 14 other lysosomal enzymes were within the range of normal control fibroblasts. The sialidase activities in fibroblasts from the patient's parents and children were 30 to 67% of normal. It is concluded that this is the first proven case of a new autosomal recessive disorder resulting in cherry-red spots, myoclonus and a sialidase deficiency.

Subarachnoid hemorrhage in rats: effect of singular or sustained hemodilution with alpha-alpha diaspirin crosslinked hemoglobin on cerebral hypoperfusion.

OBJECTIVE: To evaluate the effect of singular or sustained hemodilution, with alpha-alpha diaspirin crosslinked hemoglobin (DCLHb), on the area of hypoperfusion after subarachnoid hemorrhage. DESIGN: Prospective animal study. SETTING: Animal research laboratory. SUBJECTS: Isoflurane anesthetized, mechanically ventilated rats. INTERVENTIONS: Subarachnoid hemorrhage was induced by injecting 0.3 mL of blood into the cisterna magna. The animals were randomly assigned to one of the following groups (n = 16 in each hemodilution group; eight animals received a single treatment of hemodilution after subarachnoid hemorrhage; and, for eight animals, treatment was sustained for 48 hrs): control group (n = 8), no hematocrit (45%) manipulation; DCLHb group (n = 16), hematocrit decreased to 30% with DCLHb; or Alb group (n = 16), hematocrit decreased to 30% with human serum albumin. After 48 hrs, the area of hypoperfusion (cerebral blood flow < 40 ml/100g/min) was determined with 14C-iodoantipyrine in five coronal brain sections. MEASUREMENTS AND MAIN RESULTS: For both singular and sustained treatment, the area of hypoperfusion was less in both hemodilution groups than in the control group (p<.05). For four of the five coronal brain sections, no differences were found between the DCLHb and Alb groups within a given hemodilution protocol. In addition, in four of the five coronal brain sections for the DCLHb hemodilution groups and in all five sections for the albumin hemodilution groups, the area of hypoperfusion was less for rats that received sustained hemodilution compared with their respective groups in the singular treatment protocol (p<.05). CONCLUSIONS: These data support the hypothesis that hemodilution with molecular hemoglobin decreases hypoperfusion after subarachnoid hemorrhage and that sustained hemodilution is more effective than singular treatment. The data do not support the notion that intravascular DCLHb has an adverse effect on cerebral ischemia after subarachnoid hemorrhage.

Alterations in cultured fibroblasts of sibs with an infantile form of a free (unbound) sialic acid storage disorder.

Cultured fibroblasts from two sibs with generalized hypertonia, hepatosplenomegaly, and psychomotor retardation within the first year of life were found to have unusual morphologic features. When examined by phase microscopy, the unstained and unfixed cells contained a large number of vacuolated structures whose gross appearance resembled that of a honeycomb in the cell cytoplasm. Electron microscopy studies, following fixation, showed the "honeycombing" to be the result of numerous, closely packed, cytoplasmic, membrane-bound vacuoles. In some of these structures the remains of fibrilogranular material could be detected. Biochemical analysis of crude sonicates of these cells revealed increased levels (4--7 x N) of an acid soluble component that reacted with thiobarbituric acid. Analysis of trimethylsilyl derivatives of this material by gas liquid chromatography and mass spectrometry showed it to be indistinguishable from sialic acid (N-acetylneuraminic acid). Quantitation of this material from the cells of one of the sibs after isolation on a Dowex column yielded 39.8 nmoles of free (unbound) sialic acid per mg protein whereas normal fibroblasts had 1--2 nmoles per mg. Bound sialic acid levels were at the upper limits of normal (24.8 versus 11--23 nmoles per mg protein). The concentration of cytidine monophosphate-sialic acid was normal. After incubation of the patient's fibroblasts with [3H]-N-acetylmannosamine for 72 h, there was a 7-fold increase (compared to normal fibroblasts) in the amount of radioactivity in free sialic acid present in the acid soluble fraction. The amount of labeled, bound sialic acid in the acid-insoluble pool, however, was the same in both patient and control fibroblasts.

Impaired cerebroside sulfate hydrolysis in fibroblasts of sibs with "pseudo" arylsulfatase A deficiency without metachromatic leukodystrophy.

Low arylsulfatase A levels are reported in two siblings, one with a neurologic disability not typical for metachromatic leukodystrophy, the other a healthy 18-year-old female with a normal developmental history. In both individuals, arylsulfatase A levels in white blood cells were 7-8% of control values. Cultured fibroblasts gave low values (8-10% of normal) for both cerebroside sulfatase and arylsulfatase A activities. Other family members had enzyme levels consistent with heterozygote or normal status. Cerebroside sulfate loading tests of cultured fibroblasts in 199-CO2 media were normal for all family members who were tested. In MEM-HEPES media, however, cells from the two arylsulfatase A deficient siblings showed attenuated sulfolipid catabolism. Additional clinical and laboratory studies on these individuals failed to demonstrate any features suggestive of metachromatic leukodystrophy, i.e., normal nerve conduction velocities, normal sural nerve biopsy results, and normal urinary sulfatide excretion. It is concluded that the neurologic abnormalities in the one sibling are not the result of the low enzyme activity and that both individuals represent examples of pseudo arylsulfatase A deficiency (arylsulfatase A deficiency without metachromatic leukodystrophy). These results thus call into question the ability of the high-sensitivity cerebroside sulfate loading test as carried out in MEM-HEPES media to differentiate pathologically significant defects i.e., metachromatic leukodystrophy from benign "pseudo-deficiencies."

Nonuniform deficiency of hexosaminidase A in tissues and fluids of two unrelated individuals.

Serum samples from two unrelated, clinically normal individuals lacked detectable hexosaminidase A by heat inactivation and electrophoretic analysis. In contrast, 15 and 17% of the hexosaminidase in their leukocytes and 23 and 26% of the hexosaminidase of their cultured fibroblasts had the heat stability and electrophoretic properties of the A form of this enzyme. An in vitro measurement of fibroblasts GM2 ganglioside-beta-galactosaminidase was in the range expected for Tay-Sachs disease (TSD) heterozygotes (2.5 and 3.1 versus a normal mean of 3.7). In contrast, fibroblasts from a patient with TSD, analyzed in an identical fashion, contained no detectable activity. Ten days after addition of labeled GM2 ganglioside to the medium of the cultured fibroblasts, 43 and 59% of the radioactivity taken up by the cells of these patients remained as unhydrolyzed ganglioside as compared with 94% in TSD fibroblasts and 42% in control cells. An analysis of sphingolipid composition by high performance liquid chromatography although the endogenous level of GM2 was elevated in TSD fibroblasts (0.39 nmoles/mg protein) there was no increase in the cells of these patients (0 and 0.12 versus control of 0.17 nmoles/mg protein). Finally, the synthesis of hexosaminidase was examined by an electrophoretic analysis of immunoprecipitates of the enzyme precursors that had been radiolabeled by culturing fibroblasts in medium containing [3H]-leucine. These studies revealed a normal pattern of biosynthesis, processing and secretion of the alpha and beta chains. The ratio of the alpha chain to the beta chain, however, was in the range expected for TSD heterozygotes.

Two clonal cell populations (mosaicism) in a 46,XY male with mucolipidosis II (I-cell disease)--an autosomal recessive disorder.

Cultured fibroblasts from a 46,XY male with an atypical form of mucolipidosis II (I-cell disease) had two distinct phenotypes. One population of these fibroblasts had the morphological and biochemical features characteristic of I-cell disease, while the remaining cells were indistinguishable from normal fibroblasts. Direct evidence that the patient was a mosaic, having two cell populations, was provided by the establishment of pure, stable clones of both wild type and I-cell fibroblasts from each of two biopsies obtained several months apart. Additionally, it was shown that the I-cell fibroblasts lacked UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosaminylphosphotransferase while the morphologically normal cells contained levels of this enzyme just below or at the lower end of the normal range.