Mucolipidosis
IV
Clinical, Ultrastructural, Histochemical, of a Case, Including a Brain Biopsy
and Chemical Studies
Isabel Tellez-Nagel, MD; Isabelle Rapin, MD; Takeo Iwamoto, MD; Anne B. Johnson, MD; William T. Norton, PhD; Harold Nitowsky, MD
\s=b\A 7-year-old Ashkenazi Jewish boy with normal early development started to regress at 8 months of age and made no further developmental progress. Corneal clouding was noted at age 10 months. Corneal and conjunctival biopsy at 14 months, cerebral biopsy at 24 months, and fibroblast cultures at 32 months showed lysosomal inclusions, suggesting the storage of lipid-like and mucopolysaccharide-like material. In the brain, dense fluorescent inclusions resembled those in ceroid-lipofuscinosis. Total ganglioside content of white matter was raised, but the pattern was normal. The level of nonlipid hexosamine in the brain was normal. The cornea and conjunctiva contained electronlucent vacuoles resembling those in the mucopolysaccharidoses. Cornea, brain, and lymphocytes contained concentric membranous lamellar structures reminiscent of those in the gangliosidoses. The clinical picture and ultrastructural findings support the impression that this case belongs to a new variant of the mucolipidoses, mucolipidosis IV. (Arch Neurol 33:828-835, 1976)
publication March 15, 1976. the departments of pathology (neuropa-
Accepted From
for
thology) (Dr Johnson), medicine (Dr Tellez\x=req-\ Nagel), pediatrics (Drs Rapin and Nitowsky), the Saul R. Korey Department of Neurology (Drs Rapin and Norton), and the Rose F. Kennedy
Center for Research in Mental Retardation and Human Development (Dr Nitowsky), Albert Einstein College of Medicine, Bronx, NY, and the Eye Institute (Dr Iwamoto), Columbia University College of Physicians and Surgeons, New York. Reprint requests to Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461
(Dr Tellez-Nagel).
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progress toward
Consimucopolysaccharide derable sphingolipid lysosomal classification
a
of
and
storage disorders has resulted from
the demonstration of a specific catabolic enzymatic defect in many of these diseases.1 The mucolipidoses,5 which remain incompletely defined, have features of both the mucopolysaccharidoses and the lipidoses. Af¬ fected children do not have mucopolysacchariduria and appear to store lipid-like and polysaccharide-like jmaterial in their tissues. The prototype of this group is generalized GM1 gangliosidosis where ganglioside accumu¬ lates in the nervous system and keratan sulfate-like material in the viscera.4 Others include mucolipidoses types I (lipomucopolysaccharidosis), II (I-cell disease), and III (pseudo-Hurler polydystrophy), fucosidosis, mannosidosis, ceramidosis (Farber lipogranulomatosis), and metachromatic leukodystrophy with multiple sulfatase -
deficiency. Recently, a new variant of mucolipi¬ dosis, so-called mucolipidosis IV, was reported by Berman et al.' Within a year, three other
discov¬ ered in Israeli The parents of these four children were all Ashkenazi Jews originating from southern Poland. The children were noted to have cloudy corneas soon after birth. Pro¬ found retardation became evident by age 1 year. They are described as physically attractive, without skeletal deformity, organomegaly, gross ab¬ normality of the neurologic examinacases were
tion, or seizures. They did
not excrete
excessive amounts in the urine. Ultrastructural study of skin fibroblasts and conjunc¬ tiva suggested abnormal accumula¬ tion of both polysaccharide-like and lipid-like materials, which remain to be characterized. Prenatal diagnosis, based on the ultrastructural ap¬ pearance of cultured amniotic cells, appears possible.7 We describe here an additional example of mucolipidosis IV, includ¬ ing chemical, histochemical, and ul¬ trastructural information obtained from a cerebral biopsy.
mucopolysaccharide
in
REPORT OF A CASE A 7-year-old boy has spent the last five years in a facility for the retarded. He was born at term after an uneventful preg¬ nancy and delivery to a nonconsanguinous Ashkenazi Jewish couple aged 25 and 24 years. Their ancestors came to the United States from Warsaw, Poland, Hungary, and Austria. There is no family history of neurologic disease. The paternal grand¬ mother and her sister developed retinitis pigmentosa in middle life, but none of their forebears or descendants are known to have had this disease. An older brother, aged 8V2 years, is well. The patient was noted at birth to have a high arched palate and dysplastic pinnas. General health and somatic growth have been normal, except that the boy became microcephalic by age 4V2 years. Early motor development was normal; adaptive development was slow. He smiled at 3 months. At 8 months, he did not play or respond emotionally. At 14 months, he no longer pulled to standing. No further regression has occurred, and, at 7
Fig 1 .—Patient
at 4 years of age. No skel¬ etal or cranial abnormalities present. Facies unremarkable except for low-set ears.
about sitting on his haunches. He does not feed himself, is not toilet trained, responds to no verbal commands, has developed no speech, and does not play with objects. He localizes sound, smiles and laughs to himself, but pays little attention to his environment (Fig 1). He has had no seizures. Poor vision was suspected at 10 months, and examination showed diffuse, dense corneal clouding. This decreased sub¬ stantially by 24 months, permitting fundoscopic examination for the first time, but variable clouding has been present on all subsequent examinations. The lens is clear. The retina has an unusual sheen, years, he
moves
suggesting early tapetoretinal degenera¬ tion, but there are no clumps of pigment or macular changes. The vessels appear thin; the optic disk is pale. An electroretinogram could not be obtained, Repeated examinations have shown no skin, joint, or bony abnormality, and no organomegaly. The back is kyphotic, but there is no scoliosis or gibbus. Tone has been normal or somewhat decreased, with a tendency to scissoring when he is held upright. Tendon stretch reflexes have always been hyperactive, with clonus and upgoing toes. There is no muscular wasting. He responds to pinprick by crying. He drools excessively, but swallows without difficulty. Normal results were obtained from the following laboratory tests at 24 months: blood cell count, urinalysis, serum electro¬ lytes, fasting blood sugar, blood urea
nitrogen, serum protein electrophoresis, karyotype in cultured leukocytes, paper chromatography of the urine for aminoacids, electromyography, nerve conduction velocities, and intravenous pyelography. Transaminase levels were mildly elevated (serum glutamic oxaloacetic transaminase, 84 and 74 units; serum glutamic pyruvic transaminase, 54 units). Electrocardiogram
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Fig 2.—Top left, Neurons of cortex contain storage bodies with very slight perikaryal swelling (hematoxylin-eosin, original magnification 1,000). Top right, Neuron and satellite glia filled with densely stained granules (toluidine blue epoxy resin, original magnification 400). Bottom left, White matter oligodendrocytes contain storage matter (toluidine blue epoxy resin, original magnification 400). Bottom right, Endothelial and perithelial cells also contain storage material (toluidine blue epoxy resin, original magnification 400). was
normal, except for
a
partial right
bundle branch block. Bone marrow was normal at 14 months and 27 months, without storage cells. Skeletal survey at 32 months was not suggestive of a mucopolysaccharidosis. Electroencephalogram at 14 months was mildly slow; at 23 months, it was normal, without paroxysmal features. At 24 months, a pneumoencephalogram showed mild ventricular dilation; the cerebrospinal fluid protein content was 7 mg/ 100 ml. Twenty-four-hour excretion of mucopolysaccharides at 1, 2, and 3 years was normal. The following biopsy specimens were obtained without incident: right cornea and conjunctiva at 14 months, right frontal
lobe at 24 months, and skin for fibroblast culture at 32 months. Informed consent for each biopsy was obtained from the
parents. MATERIALS AND METHODS Brain Biopsy A
of frontal cortex with a few millimeters of subcortical white matter was obtained. Fixation and staining meth¬ ods for histology, enzyme histochemistry, and electron microscopy have been detailed elsewhere.8
sample
Histology, Histochemistry, and Electron sections were Microscopy.—Paraffin stained by classic neuropathological pro¬ cedures. To study the properties of the
Storage material in brain cells, frozen sections and cryostat sections postfixed in
10% formaldehyde solution were stained with PAS, oil red 0, and Sudan black with and without a ten-minute pretreat¬ ment with ethanol or acetone. Some fixed and unfixed cryostat sections were treated with diastase or extracted with acid-alco¬ hol, ether, chloroform or chloroformmethanol (2:1) for 30 minutes and for 8 days at room temperature, then fixed in a 10% formaldehyde solution and stained with PAS. Metachromasia was studied in Alcian blue, crystal violet (0.1% at pH 2.8), and 0.1% aqueous toluidine blue prepara¬ tions. In addition, cryostat sections were stained with 0.05% Alcian blue at pH 5.7 with both 0.1M and 0.3M MgCl2 to detect mucopolysaccharides.9 Autofluorescence was studied in deparaffinized sections fixed in a 10% formaldehyde solution. A mercury ultraviolet light source was used with filters 47 and 50. The emission spec¬ trum was measured at a wavelength of 470 Angstroms. Acid phosphatase activity was studied in frozen sections, fixed in a 10% formaldehyde solution and glutaraldehyde, by the Gomori method and examined with the light and electron microscopes. For electron microscopy, gray and white mat¬ ter were fixed in 5% glutaraldehyde in 0.067M phosphate buffer (pH 7.35) with 5% sucrose, postfixed in Dalton solution,10 and 1-jum sections were stained with toluidine blue. Ultrathin sections from selected blocks were studied by electron micros¬ copy. Chemical Analyses.—A portion of the biopsy sample was dissected into gray (287.0 mg) and white matter (55.2 mg), immediately frozen in dry ice, and stored at —90 C until analyzed. Extraction of the tissue and determination of water, insol¬ uble residue, upper phase weight, total lipid content, and individual lipid contents carried out as previously de¬ were scribed.11 12 Analysis of ganglioside Nacetylneuraminic acid (NANA) was per¬ formed by gas-liquid chromatography.13 Cerebrosides and sulfatides were sepa¬ rated on a silicagel high purity plate, using the solvent system CHC13-CH30H-H20, 70:30:4 (v/v/v). The zones were visualized by iodine vapor, scraped off, and assayed a
quantitatively by gas-liquid chromatogra¬ phy.14 For the determination of nonlipid hexosamine, 10 mg of the lipid-free insol¬ uble residue was hydrolyzed with 2 ml of IN HC1 in a sealed, evacuated ampule at 10 C for 15 hours. The hydrolysate was filtered, dried, and assayed for hexosamine by the Elson-Morgan reaction.15 Ocular Tissue Tissue from the
peripheral
cornea
and
Downloaded From: http://archneur.jamanetwork.com/ by a University of Iowa User on 06/18/2015
Fig 3.—Cortical neuron with perikaryon occupied by bodies (original magnification 20,250).
conjunctiva was obtained surgically by superficial keratectomy and conjunctival biopsy. Half of the tissue was processed for electron microscopy.16 The other half was fixed in absolute alcohol, processed for histochemistry, and stained with Alcian blue, toluidine blue, and PAS. Cultured Skin Fibroblasts derived from a skin biopsy specimen obtained without anesthesia, using a high-speed electric rotating biopsy punch with a core drill 3 mm in diameter.17 The core of skin was minced into 20 or 30 fragments and cultured in McCoy 5A medium with 20% fetal calf serum at 37 C in an atmosphere of 5% C02 in air. After the first subculture, the cells were transferred in replicate to flasks. After seven to eight days, the confluent monolayer of fibroblast cells was dispersed with 0.05% trypsin containing 0.2% edetic acid in calcium and magneA cell culture strain
was
numerous
compound storage
sium-free Hank solution, and transferred to fresh medium for determination of enzyme activity. Washed cell suspensions were stored in 0.25M sucrose at —70 C for as long as three to four weeks prior to assay. Preliminary studies showed no decrease in enzyme activity with storage for this duration. Cell-free extracts were prepared by sonic vibration of thawed cell suspensions to which 0.1% octoxynol-9 (Triton X-100) was added (three parts cell to one part octoxynol-9) for one minute, using an ice water-cooled oscillator. Assays were carried out within one hour of sonication. The amount of a given glycosidase activity at pH 4.3 in the cell sonicate was determined by a modification of the proce¬ dure of Bosmann,18 using the following substrates: jo-nitrophenyl-iV-acetyl-/?-
D-glucosaminide, p-nitrophenyl-a-D-glucoside, p-nitrophenyl-a-D-galactoside, jö-nitrophenyl-ß-D-glucoside, p-nitro-
Fig 4.—Most common type of storage body (compound bodies). Compact lamellae with 50- to 60-A periodicity form the periphery and slowly blend into granular matrix that occasionally has fine crystalline pattern (original magnification 75,500). Table 1.—Chemical
Composition
of
Gray
5.—Different types of inclusion bodies in oligodendrocytes formed by very densely packed lamellae, suggesting sometimes a
Fig
fingerprint pattern (original magnification
Controls*
Water, % dry weight
CHCI3-CH3OH insoluble residue, % dry weight Upper phase solids, % dry weight Total lipidu content, /o dry uuuieiii, % uiy weight weiyiu Nonlipid hexosamine, ng/ng insoluble residue Ganglioside NANA, ng/gm wet weight Weiaht
IV
Clinical, Ultrastructural, Histochemical, of a Case, Including a Brain Biopsy
and Chemical Studies
Isabel Tellez-Nagel, MD; Isabelle Rapin, MD; Takeo Iwamoto, MD; Anne B. Johnson, MD; William T. Norton, PhD; Harold Nitowsky, MD
\s=b\A 7-year-old Ashkenazi Jewish boy with normal early development started to regress at 8 months of age and made no further developmental progress. Corneal clouding was noted at age 10 months. Corneal and conjunctival biopsy at 14 months, cerebral biopsy at 24 months, and fibroblast cultures at 32 months showed lysosomal inclusions, suggesting the storage of lipid-like and mucopolysaccharide-like material. In the brain, dense fluorescent inclusions resembled those in ceroid-lipofuscinosis. Total ganglioside content of white matter was raised, but the pattern was normal. The level of nonlipid hexosamine in the brain was normal. The cornea and conjunctiva contained electronlucent vacuoles resembling those in the mucopolysaccharidoses. Cornea, brain, and lymphocytes contained concentric membranous lamellar structures reminiscent of those in the gangliosidoses. The clinical picture and ultrastructural findings support the impression that this case belongs to a new variant of the mucolipidoses, mucolipidosis IV. (Arch Neurol 33:828-835, 1976)
publication March 15, 1976. the departments of pathology (neuropa-
Accepted From
for
thology) (Dr Johnson), medicine (Dr Tellez\x=req-\ Nagel), pediatrics (Drs Rapin and Nitowsky), the Saul R. Korey Department of Neurology (Drs Rapin and Norton), and the Rose F. Kennedy
Center for Research in Mental Retardation and Human Development (Dr Nitowsky), Albert Einstein College of Medicine, Bronx, NY, and the Eye Institute (Dr Iwamoto), Columbia University College of Physicians and Surgeons, New York. Reprint requests to Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461
(Dr Tellez-Nagel).
Downloaded From: http://archneur.jamanetwork.com/ by a University of Iowa User on 06/18/2015
progress toward
Consimucopolysaccharide derable sphingolipid lysosomal classification
a
of
and
storage disorders has resulted from
the demonstration of a specific catabolic enzymatic defect in many of these diseases.1 The mucolipidoses,5 which remain incompletely defined, have features of both the mucopolysaccharidoses and the lipidoses. Af¬ fected children do not have mucopolysacchariduria and appear to store lipid-like and polysaccharide-like jmaterial in their tissues. The prototype of this group is generalized GM1 gangliosidosis where ganglioside accumu¬ lates in the nervous system and keratan sulfate-like material in the viscera.4 Others include mucolipidoses types I (lipomucopolysaccharidosis), II (I-cell disease), and III (pseudo-Hurler polydystrophy), fucosidosis, mannosidosis, ceramidosis (Farber lipogranulomatosis), and metachromatic leukodystrophy with multiple sulfatase -
deficiency. Recently, a new variant of mucolipi¬ dosis, so-called mucolipidosis IV, was reported by Berman et al.' Within a year, three other
discov¬ ered in Israeli The parents of these four children were all Ashkenazi Jews originating from southern Poland. The children were noted to have cloudy corneas soon after birth. Pro¬ found retardation became evident by age 1 year. They are described as physically attractive, without skeletal deformity, organomegaly, gross ab¬ normality of the neurologic examinacases were
tion, or seizures. They did
not excrete
excessive amounts in the urine. Ultrastructural study of skin fibroblasts and conjunc¬ tiva suggested abnormal accumula¬ tion of both polysaccharide-like and lipid-like materials, which remain to be characterized. Prenatal diagnosis, based on the ultrastructural ap¬ pearance of cultured amniotic cells, appears possible.7 We describe here an additional example of mucolipidosis IV, includ¬ ing chemical, histochemical, and ul¬ trastructural information obtained from a cerebral biopsy.
mucopolysaccharide
in
REPORT OF A CASE A 7-year-old boy has spent the last five years in a facility for the retarded. He was born at term after an uneventful preg¬ nancy and delivery to a nonconsanguinous Ashkenazi Jewish couple aged 25 and 24 years. Their ancestors came to the United States from Warsaw, Poland, Hungary, and Austria. There is no family history of neurologic disease. The paternal grand¬ mother and her sister developed retinitis pigmentosa in middle life, but none of their forebears or descendants are known to have had this disease. An older brother, aged 8V2 years, is well. The patient was noted at birth to have a high arched palate and dysplastic pinnas. General health and somatic growth have been normal, except that the boy became microcephalic by age 4V2 years. Early motor development was normal; adaptive development was slow. He smiled at 3 months. At 8 months, he did not play or respond emotionally. At 14 months, he no longer pulled to standing. No further regression has occurred, and, at 7
Fig 1 .—Patient
at 4 years of age. No skel¬ etal or cranial abnormalities present. Facies unremarkable except for low-set ears.
about sitting on his haunches. He does not feed himself, is not toilet trained, responds to no verbal commands, has developed no speech, and does not play with objects. He localizes sound, smiles and laughs to himself, but pays little attention to his environment (Fig 1). He has had no seizures. Poor vision was suspected at 10 months, and examination showed diffuse, dense corneal clouding. This decreased sub¬ stantially by 24 months, permitting fundoscopic examination for the first time, but variable clouding has been present on all subsequent examinations. The lens is clear. The retina has an unusual sheen, years, he
moves
suggesting early tapetoretinal degenera¬ tion, but there are no clumps of pigment or macular changes. The vessels appear thin; the optic disk is pale. An electroretinogram could not be obtained, Repeated examinations have shown no skin, joint, or bony abnormality, and no organomegaly. The back is kyphotic, but there is no scoliosis or gibbus. Tone has been normal or somewhat decreased, with a tendency to scissoring when he is held upright. Tendon stretch reflexes have always been hyperactive, with clonus and upgoing toes. There is no muscular wasting. He responds to pinprick by crying. He drools excessively, but swallows without difficulty. Normal results were obtained from the following laboratory tests at 24 months: blood cell count, urinalysis, serum electro¬ lytes, fasting blood sugar, blood urea
nitrogen, serum protein electrophoresis, karyotype in cultured leukocytes, paper chromatography of the urine for aminoacids, electromyography, nerve conduction velocities, and intravenous pyelography. Transaminase levels were mildly elevated (serum glutamic oxaloacetic transaminase, 84 and 74 units; serum glutamic pyruvic transaminase, 54 units). Electrocardiogram
Downloaded From: http://archneur.jamanetwork.com/ by a University of Iowa User on 06/18/2015
Fig 2.—Top left, Neurons of cortex contain storage bodies with very slight perikaryal swelling (hematoxylin-eosin, original magnification 1,000). Top right, Neuron and satellite glia filled with densely stained granules (toluidine blue epoxy resin, original magnification 400). Bottom left, White matter oligodendrocytes contain storage matter (toluidine blue epoxy resin, original magnification 400). Bottom right, Endothelial and perithelial cells also contain storage material (toluidine blue epoxy resin, original magnification 400). was
normal, except for
a
partial right
bundle branch block. Bone marrow was normal at 14 months and 27 months, without storage cells. Skeletal survey at 32 months was not suggestive of a mucopolysaccharidosis. Electroencephalogram at 14 months was mildly slow; at 23 months, it was normal, without paroxysmal features. At 24 months, a pneumoencephalogram showed mild ventricular dilation; the cerebrospinal fluid protein content was 7 mg/ 100 ml. Twenty-four-hour excretion of mucopolysaccharides at 1, 2, and 3 years was normal. The following biopsy specimens were obtained without incident: right cornea and conjunctiva at 14 months, right frontal
lobe at 24 months, and skin for fibroblast culture at 32 months. Informed consent for each biopsy was obtained from the
parents. MATERIALS AND METHODS Brain Biopsy A
of frontal cortex with a few millimeters of subcortical white matter was obtained. Fixation and staining meth¬ ods for histology, enzyme histochemistry, and electron microscopy have been detailed elsewhere.8
sample
Histology, Histochemistry, and Electron sections were Microscopy.—Paraffin stained by classic neuropathological pro¬ cedures. To study the properties of the
Storage material in brain cells, frozen sections and cryostat sections postfixed in
10% formaldehyde solution were stained with PAS, oil red 0, and Sudan black with and without a ten-minute pretreat¬ ment with ethanol or acetone. Some fixed and unfixed cryostat sections were treated with diastase or extracted with acid-alco¬ hol, ether, chloroform or chloroformmethanol (2:1) for 30 minutes and for 8 days at room temperature, then fixed in a 10% formaldehyde solution and stained with PAS. Metachromasia was studied in Alcian blue, crystal violet (0.1% at pH 2.8), and 0.1% aqueous toluidine blue prepara¬ tions. In addition, cryostat sections were stained with 0.05% Alcian blue at pH 5.7 with both 0.1M and 0.3M MgCl2 to detect mucopolysaccharides.9 Autofluorescence was studied in deparaffinized sections fixed in a 10% formaldehyde solution. A mercury ultraviolet light source was used with filters 47 and 50. The emission spec¬ trum was measured at a wavelength of 470 Angstroms. Acid phosphatase activity was studied in frozen sections, fixed in a 10% formaldehyde solution and glutaraldehyde, by the Gomori method and examined with the light and electron microscopes. For electron microscopy, gray and white mat¬ ter were fixed in 5% glutaraldehyde in 0.067M phosphate buffer (pH 7.35) with 5% sucrose, postfixed in Dalton solution,10 and 1-jum sections were stained with toluidine blue. Ultrathin sections from selected blocks were studied by electron micros¬ copy. Chemical Analyses.—A portion of the biopsy sample was dissected into gray (287.0 mg) and white matter (55.2 mg), immediately frozen in dry ice, and stored at —90 C until analyzed. Extraction of the tissue and determination of water, insol¬ uble residue, upper phase weight, total lipid content, and individual lipid contents carried out as previously de¬ were scribed.11 12 Analysis of ganglioside Nacetylneuraminic acid (NANA) was per¬ formed by gas-liquid chromatography.13 Cerebrosides and sulfatides were sepa¬ rated on a silicagel high purity plate, using the solvent system CHC13-CH30H-H20, 70:30:4 (v/v/v). The zones were visualized by iodine vapor, scraped off, and assayed a
quantitatively by gas-liquid chromatogra¬ phy.14 For the determination of nonlipid hexosamine, 10 mg of the lipid-free insol¬ uble residue was hydrolyzed with 2 ml of IN HC1 in a sealed, evacuated ampule at 10 C for 15 hours. The hydrolysate was filtered, dried, and assayed for hexosamine by the Elson-Morgan reaction.15 Ocular Tissue Tissue from the
peripheral
cornea
and
Downloaded From: http://archneur.jamanetwork.com/ by a University of Iowa User on 06/18/2015
Fig 3.—Cortical neuron with perikaryon occupied by bodies (original magnification 20,250).
conjunctiva was obtained surgically by superficial keratectomy and conjunctival biopsy. Half of the tissue was processed for electron microscopy.16 The other half was fixed in absolute alcohol, processed for histochemistry, and stained with Alcian blue, toluidine blue, and PAS. Cultured Skin Fibroblasts derived from a skin biopsy specimen obtained without anesthesia, using a high-speed electric rotating biopsy punch with a core drill 3 mm in diameter.17 The core of skin was minced into 20 or 30 fragments and cultured in McCoy 5A medium with 20% fetal calf serum at 37 C in an atmosphere of 5% C02 in air. After the first subculture, the cells were transferred in replicate to flasks. After seven to eight days, the confluent monolayer of fibroblast cells was dispersed with 0.05% trypsin containing 0.2% edetic acid in calcium and magneA cell culture strain
was
numerous
compound storage
sium-free Hank solution, and transferred to fresh medium for determination of enzyme activity. Washed cell suspensions were stored in 0.25M sucrose at —70 C for as long as three to four weeks prior to assay. Preliminary studies showed no decrease in enzyme activity with storage for this duration. Cell-free extracts were prepared by sonic vibration of thawed cell suspensions to which 0.1% octoxynol-9 (Triton X-100) was added (three parts cell to one part octoxynol-9) for one minute, using an ice water-cooled oscillator. Assays were carried out within one hour of sonication. The amount of a given glycosidase activity at pH 4.3 in the cell sonicate was determined by a modification of the proce¬ dure of Bosmann,18 using the following substrates: jo-nitrophenyl-iV-acetyl-/?-
D-glucosaminide, p-nitrophenyl-a-D-glucoside, p-nitrophenyl-a-D-galactoside, jö-nitrophenyl-ß-D-glucoside, p-nitro-
Fig 4.—Most common type of storage body (compound bodies). Compact lamellae with 50- to 60-A periodicity form the periphery and slowly blend into granular matrix that occasionally has fine crystalline pattern (original magnification 75,500). Table 1.—Chemical
Composition
of
Gray
5.—Different types of inclusion bodies in oligodendrocytes formed by very densely packed lamellae, suggesting sometimes a
Fig
fingerprint pattern (original magnification
Controls*
Water, % dry weight
CHCI3-CH3OH insoluble residue, % dry weight Upper phase solids, % dry weight Total lipidu content, /o dry uuuieiii, % uiy weight weiyiu Nonlipid hexosamine, ng/ng insoluble residue Ganglioside NANA, ng/gm wet weight Weiaht
