Timothy A. Lyerla

GM2-ganglioside metabolism in cultured human skin fibroblasts: unambiguous diagnosis of GM2-gangliosidosis

The metabolism of GM2-ganglioside was studied in situ using cultured skin fibroblasts from normal individuals and patients with different forms of GM2-gangliosidosis. [3H]Sphingosine-labeled GM2 was provided in the culture medium to confluent cells in 6-cm petri dishes. After 10 days, the cells were washed free of radioactivity and harvested by trypsinization. The cellular lipids were extracted and analyzed for radioactivity in GM2 and its metabolic products. In fibroblasts from healthy subjects, 50-60% of the total cellular radioactivity was found in the neutral glycosphingolipids, ceramide, sphingomyelin and fatty acids. Degradation of the labeled GM2 progressed rapidly via GM3, ceramide dihexoside and ceramide monohexoside with a build-up of radioactivity mainly in the ceramide pool of the cell. The labeled ceramide is also reutilized for the synthesis of ceramide trihexoside, globoside and sphingomyelin or is converted to fatty acid and incorporated in ester linkages. In contrast, cells from patients with GM2-gangliosidosis representing Tay-Sachs, Sandhoff and AB variant forms of the disease did not metabolize the ingested labeled GM2-like controls. Nearly all of the radioactivity was present in the ganglioside fraction in the lipid extracts from these cells and consisted of unhydrolyzed GM2. High-performance liquid chromatographic analysis of monosialogangliosides from cells grown without added labeled GM2 in the medium indicated accumulation of endogenously synthesized GM2 in cell lines from all patients with GM2 gangliosidosis compared to healthy controls. This approach provides a reliable tool for pre- and post-natal diagnosis of all forms of GM2-gangliosidosis without ambiguity.

The accumulation and metabolism of glycosphingolipids in primary kidney cell cultures from beige mice

In the normal C57BL/6J male mouse a specific subset of the kidney glycosphingolipids which is associated with multilamellar bodies of lysosomal origin and represents about 10% of the total kidney glycolipids, is excreted into the urine each day. This excretion is blocked and glycosphingolipids accumulate in the kidney of bgJ/bgJ mutants of this strain. To examine this process in vitro, glycosphingolipid metabolism and excretion were studied in beige mouse kidney cell cultures. Primary kidney cell cultures from male C57BL/6J control and bgJ/bgJ beige mutants were grown in D-valine medium and glycosphingolipids labeled with [3H]palmitate. As we have shown previously, the giant lysosomes of altered morphology were maintained in cultures of the beige kidney cells. Beige-J and control cells synthesized the same types of glycosphingolipids, but the mutant cells had quantitatively higher levels of these compounds than control cells, as determined by high performance liquid chromatography. Beige-J cells incorporated more [3H]palmitate into glycospingholipids than control cells on a cpm/mg protein basis and the specific activity (cpm/pmole glycosphingolipid) was lower in beige cells. Medium from beige-J cells accumulated more glycosphingolipids than that from control cells in a 24 h period. The glycosphingolipids released into the medium as determined by HPLC were primarily non-lysosomal types and both control and mutant cells retained the glycosphingolipids associated with lysosomal multilamellar bodies excreted in vivo. The elevated levels of lysosomal glycosphingolipids and the dysmorphic lysosomes in primary cultures of beige cells, then, are not caused by a mutant block in secretion of lysosomes. (Mol Cell Biochem 118: 61–66, 1992)

EXPRESSION OF GLYCOSPHINGOLIPIDS IN SERUM-FREE PRIMARY CULTURES OF MOUSE KIDNEY CELLS : MALE-FEMALE DIFFERENCES AND ANDROGEN SENSITIVITY

The expression of neutral glycosphingolipids was examined in primary kidney cell cultures derived from adult male and female beige mutant mice (C57BL/6J;bgj/bgj) with enrichment for proximal tubule cells during preparation of explants and using defined serum-free medium for the culture conditions. Cell proliferated for 7 daysin vitro to provide confluent or nearly confluent monolayers of epithelial-type growth indicative of proximal tubule cells. The malevs female differences in neutral glycosphingolipids seen in the kidneyin vivo were retained in these 7 day cultures. Cultures derived from males contained galacto- and digalactosylceramides whereas those from females did not express these types of glycolipids. Also, male cells had higher ratios of sphingosine: phytosphingosine containing species in Nfa (non-hydroxy fatty acid) globotriaosylceramide and in glucosylceramide than females. The shift in sphingosine: phytosphingosine to male ratios in Nfa globotriaosylceramide and in glucosylceramide could be stimulated in female kidney cells by treatment with 10−5 M testosterone or 5α-dihydrotestosterone. The male-specific expression of neutral glycosphingolipids, then, appears to be stable character of male-type differentiation in mouse kidney that is passed on during proliferation in culture. Female kidney cells retain an ability to respond to androgens with specific changes in neutral glycosphingolipid expression during 7 days of growthin vitro in serum-free conditions, but do not respond with the induction of the male-specific glycolipids galacto-and digalactosylceramides as seenin vivo.

Alcohol dehydrogenase isozymes in the clawed frog, Xenopus laevis

Alcohol dehydrogenase (ADH; EC 1.1.1.1) activity in Xenopus laevis was highest in liver tissue, with decreasing activities in kidney, heart, and gut tissues, respectively. Essentially no activity was found among other tissues screened, including lung, ovary, eye, and testes. Also, there was no apparent sexual dimorphism of ADH activity in either liver or kidney tissue. All ADH isozymes were inhibited by 10mm pyrazole, and no eye-specific retinol dehydrogenase activity was detected on starch gel electropherograms. Isozyme patterns from 418 offspring from 11 different crosses could be explained genetically assuming the presence of two structural genes coding for ADH production: one carrying two electrophoretically separable variants and the other showing quantitative variation in its expression. The ADH system in X. laevis should be useful for studies concerning the molecular mechanisms governing the expression of ADH activity in vertebrate development.

Biochemical and morphological characterization of primary kidney cell cultures from beige mutant mice.

SummaryPrimary kidney cultures from adult beige-J (bgJ/ bgJ) mice were selected for epithelial cell growth using D-valine medium. After 2 weeks of attachment and proliferation in vitro, the cells form a confluent or nearly confluent monolayer that retains several phenotypic characteristics of the beige-J mutant. These include large, multilamellar inclusion bodies that are apparently dysmorphic lysosomes, and higher concentrations of neutral glycosphingolipids and dolichols than control cells. β-Glucuronidase activity, used as a lysosomal enzyme marker, is not elevated in beige-J-cultured kidney cells compared with controls, as it is in the intact kidney. The high levels of β-glucuronidase activity in both control and mutant cells may mask expression of this difference in vitro. The action of the beige-J mutation in kidney cells is thought to be due to a block in exocytosis that results in the accumulation of abnormal lysosomes and their components. The maintenance of the beige phenotype in vitro indicates that the mutation is not suppressed in primary kidney cell cultures. The expression of the beige phenotype in vitro should be useful for studies concerning the primary lesion of this mutation.

PIGMENTATION IN THE ORANGE TUNICATE, ECTEINASCIDIA TURBINATA

Some chemical properties of the orange pigment from eggs and embryos and the mantle tissue of adult Ecteinascidia turbinata, as well as ultrastructural characteristics of pigment masses in the mantle, have been examined. The diffuse egg and embryo pigment has solubility and absorbence characteristics of carotenoids, which are solubilized within yolk platelets. The granular mantle pigment, however, is not carotenoid. It is soluble only in organic-aqueous systems, stable to boiling and gross changes in pH, and absorbs strongly in the UV region with a single major peak at 360 nm. Mantle pigment is found in pigment bodies which ultrastructurally are composed of apparently straight and continuous tubular subunits, approximately 500 to 600 Å in diameter, and oriented parallel to the long axis of the body. The pigment bodies are not localized in obvious cells but found within large areas of fibrillar material that are bound by cytoplasmic membranes and occasionally contain an apparently degenerate nucleus. It may be that mantle pigment is derived from pigment cells of the blood which lose their typical cellular appearance as they become permanent features of the mantle tissue.

Cellular expression of the beige mouse mutation and its correction in hybrids with control human fibroblasts

SummaryFibroblasts from a beige mouse (C57BL/6J;bgJ bgJ) have been established and maintained in culture for more than 3 yr. At early passages, the mutant cells were distinguishable from C57BL/6J control mouse fibroblasts at the ultrastructural level by the presence of enlarged cytoplasmic granules. After continuous passaging, this distinguishing feature was lost from the mutant cells, correlated with their increased growth rate. Clustered, perinuclear distribution of lysosomes was retained, however, and was quantitatively different at any passage number of the beige cell line from the dispersed distribution of these organelles in control mouse fibroblasts, as analyzed by computer-aided, video-enhanced light microscopy. In somatic cell hybrids between the established beige cell line and a control human diploid fibroblast cell strain, seven uncorrected hybrid lines retained a lysosomal dispersion pattern statistically indistinguishable from that of the beige mouse cell lines. Three corrected hybrid lines had lysosomal dispersion patterns that were significantly different from the beige parent line and indistinguishable from that of the control mouse fibroblast line. Thus, lysosomal dispersion can be used objectively and quantitatively to distinguish mutant beige and control mouse fibroblasts and corrected vs. uncorrected cell hybrids made from the beige/control human somatic cell crosses.

Establishment of a galactocerebrosidase-deficient twitcher mouse cell line that expresses galactocerebrosidase activity in hybrids with control human fibroblasts.

SummaryPrimary cell cultures from twitcher (galactocerebrosidase deficient) mice were made by enzymatic dispersion and explantation of skin obtained from 3-d-old littermates of atwi+/twi×twi+/twi mating. Galactocerebrosidase activity remained deficient for two twitcher cell lines, TM-1 and TM-2, and both lines demonstrated an initial period of growth decline, followed by accelerated growth. The TM-2 line has been subcultured for more than 3.5 yr, has a modal chromosome number of 63, a doubling time of approximately 16 h, and has remained galactocerebrosidase deficient throughout its life span. These data indicate this to be an established twitcher cell line that can be continuously maintained in culture as a transformed galactocerebrosidase-deficient mouse cell line. This established line was rendered 6-thioguanine resistant so that the cells could be fused with control human fibroblasts and selected for hybrid lines in hypoxanthine-aminopterin-thymidine medium. Also, the established twitcher cells were crossed with neomycin-resistant control human fibroblasts and selected in G418 medium. Several of the hybrid lines from both crosses had higher than deficient levels of galactocerebrosidase activity initially, followed by a decrease to twitcher levels during subculture, whereas other lines retained high levels of activity. These results indicate that twitcher-human somatic cell hybrids will express galactocerebrosidase activity and thus may be useful for determining the human chromosome or chromosomes associated with this expression.

Malate dehydrogenase isozyme patterns in species of terrestrial isopods

Abstract 1. 1. Populations of nine species within four different families of terrestrial isopods (Order Oniscoidea) were examined with respect to their malate dehydrogenase (Mdh) isozyme patterns using horizontal starch gel electrophoresis. 2. 2. Cytoplasmic Mdh (Mdh-1) was anodal to mitochondrial Mdh (Mdh-2) in seven of the nine species, and the distance between the two forms was greatest in the more terrestrially adapted species. 3. 3. Mdh-2 migrated to the same position in seven, or perhaps eight, of the nine species and was monomorphic. 4. 4. Banding patterns were specific at the familial level, but within the Porcellionidae, two genera (Trachelipus and Cylisticus) had the same pattern, and the three Porcellio species studied had identical patterns. 5. 5. Mdh banding patterns appear to be useful biochemical markers for studying possible taxonomic relationships among the Oniscoidea above the species level.

Xanthine dehydrogenase activity in the clawed frog, Xenopus laevis

Xanthine dehydrogenase (XDH; EC 1.2.1.37) activity in the clawed frog, Xenopus laevis, was detected in kidney tissue homogenates, but not in skin, liver, ovaries or gut tissues. The enzyme migrated as a single band of activity on both polyacrylamide and starch gel electropherograms, exhibited substrate inhibition, and did not appear developmentally until feeding larval stages. The tissue specificity, post-fertilization stage of appearance and single isozymic form make this a useful enzyme marker for further study concerning its developmental appearance and maintenance as a kidney-specific protein.