Influence of sialic acid on cell surface properties in I-cell disease fibroblasts

Summary Fibroblasts derived from patients with I-cell disease have been shown to accumulate many natural substrates including a three to fourfold increase in sialic acid content compared to that found in normal fibroblasts. This diverse accumulation of storage material is due to a massive deficiency of multiple lysosomal hydrolases as they are preferentially excreted into the culture fluid. There is evidence that the I-cell plasma membrane itself is abnormal with respect to certain transferase activities and in its sensitivity to freezing and Triton X-100. In this study, we have shown that a neuraminidase-sensitive substrate, and perhaps others in I-cell fibroblasts, contribute to an increased electronegativity of the I-cell fibroblast surface and to the cells' sensitivity to freezing. We also found that neuraminidase treatment of I-cell fibroblasts before preservative freezing in liquid nitrogen enables the cells to adapt more easily to subculture upon thawing. This project was supported in part by National Institutes of Health (NIH) BRSG Grant RR-05493, NIH Grant 1-R01-HD-11453-01-A1, National Science Foundation Grant PCM 77-05733, and Maternal and Child Health Service Project 417. Georgirene D. Vladutiu is the recipient of Research Career Development Award 1K04 HD 00312-01A1 from the National Institutes of Health.     

Huntington's disease: the challenge for cell biologists

Huntington's disease (HD) is one of eight inherited neurodegenerative diseases caused by expansions of (CAG)(n) tracts that encode polyglutamine segments in expressed proteins. Studies of pathogenic mechanisms for all these late-onset diseases suffer from a common drawback: experimental studies require massive acceleration of a process that, in affected humans, usually takes decades. But is the rapid-onset disease of transgenic mouse models and in cells the same as the slow-onset disease in humans? We review recent work on HD, noting several issues whose significance is likely to be crucial - but which are as yet unresolved. We discuss these in light of the distinction between disease-specific pathogenic mechanisms and artifacts of polyglutamine overexpression. We suggest that the initial stages of HD result from dysfunction rather than death, and we consider the potential discovery of compounds that might interfere with early pathogenic events.     

Further evidence for a cell surface proteinase essential to the growth of cultured fibroblasts

Specific antibodies and protein proteinase inhibitors will inhibit cell-surface proteinase activity on human fibroblasts and cause a concomitant inhibition of DNA synthesis and of cell multiplication. An insolubilized proteinase inhibitor also inhibits cell multiplication. The same reagents partially inhibit the multiplication of mouse L cells, both in monolayer and suspension culture, and inhibit the mitogenic effect of epidermal growth factor (EGF) on both types of cell.     

Reduction of glyceraldehyde-3-phosphate dehydrogenase activity in Alzheimer's disease and in Huntington's disease fibroblasts

New functions have been identified for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) including its role in neurodegenerative disease and in apoptosis. GAPDH binds specifically to proteins implicated in the pathogenesis of a variety of neurodegenerative disorders including the beta-amyloid precursor protein and the huntingtin protein. However, the pathophysiological significance of such interactions is unknown. In accordance with published data, our initial results indicated there was no measurable difference in GAPDH glycolytic activity in crude whole-cell sonicates of Alzheimer's and Huntington's disease fibroblasts. However, subcellular-specific GAPDH-protein interactions resulting in diminution of GAPDH glycolytic activity may be disrupted or masked in whole-cell preparations. For that reason, we examined GAPDH glycolytic activity as well as GAPDH-protein distribution as a function of its subcellular localization in 12 separate cell strains. We now report evidence of an impairment of GAPDH glycolytic function in Alzheimer's and Huntington's disease subcellular fractions despite unchanged gene expression. In the postnuclear fraction, GAPDH was 27% less glycolytically active in Alzheimer's cells as compared with age-matched controls. In the nuclear fraction, deficits of 27% and 33% in GAPDH function were observed in Alzheimer's and Huntington's disease, respectively. This evidence supports a functional role for GAPDH in neurodegenerative diseases. The possibility is considered that GAPDH:neuronal protein interaction may affect its functional diversity including energy production and as well as its role in apoptosis.     

Qualitative and quantitative comparison of brain proteins in Alzheimer's disease

In human brain extracts, most proteins of pathological interest in Alzheimer's disease are insoluble and their analysis is often performed on denatured and reduced samples by immunoblotting after electrophoresis on polyacrylamide gel in presence of sodium dodecyl sulfate. Because we needed to accurately compare the concentration of several proteins in brain extracts to investigate the etiology of the disease, the quantitative aspect of immunoblotting was assessed and the results compared for a soluble component with those obtained by electroimmunoassay. Glial fibrillary acidic protein (GFAP) and Tau proteins were analysed by immunoblotting in brain homogenates treated with the Laemmli sample buffer from 10 control and 25 Alzheimer's disease brains. The linearity of densitometric measures of dilutions for one given sample was demonstrated. A 8 to 16-fold GFAP increase in Alzheimer brain was established. With regard to Tau proteins it was possible to show the presence of two pathological Tau variants (Tau 64 and 69) in all the Alzheimer brain homogenates, furthermore, the amount of Tau 64 and 69 was proportional to the presence of neurofibrillary degeneration. As far as alpha 1-antichymotrypsin is concerned, we showed, in a second set of brain samples (14 control and 12 Alzheimer brains), discrepancies between the results obtained by immunoblotting and by electroimmunoassay while for a given sample linearity of immunoblotting measures of dilutions of this sample was demonstrated. Quantitation by immunoblotting of such components which can be quantified using other procedures is uncertain whereas the interest of immunoblotting is undoubted for the insoluble proteins in the brain extracts.     

Molecular properties of a major cell surface protein from chick embryo fibroblasts.

The molecular structure of chick embryo fibroblast cell surface protein has been investigated by ultracentrifugation, circular dichroism, and fluorescence. Most measurements were restricted to alkaline solutions because of the limited solubility of this protein at more neutral pH values. A very high frictional ratio for the protein suggests an asymmetric structure. However, there are elements of organized structure since typical thermal transition curves were found by several methods. Consequently, a model in which ordered domains are connected by flexible polypeptide chains seems to account for all the hydrodynamic and optical data.     

Mechanisms of neuronal cell death in Huntington's disease

Huntington's disease (HD) is a genetically dominant neurodegenerative condition caused by an unique mutation in the disease gene huntingtin. Although the Huntington protein (Htt) is ubiquitously expressed, expansion of the polyglutamine tract in Htt leads to the progressive loss of specific neuronal subpopulations in HD brains. In this article, we will summarize the current understanding on mechanisms of how mutant Htt can elicit cytotoxicity, as well as how the selective sets of neuronal cell death occur in HD brains.     

Characterization of the cell kinetics and growth properties of cystic fibrosis diploid fibroblasts in vitro.

The population growth kinetics of human diploid skin fibroblasts derived from cystic fibrosis homozygotes and heterozygotes and from normal subjects were investigated. Our data suggest the following: 1. Population doubling times increase with time in culture, with no significant differences observed among the three genotypes tested, when data were compared at the same subculture times or phases of growth. 2. The fraction of dividing cells in a population decreased with the duration in which the cells were in culture. No significant differences were obtained for cells derived from the three genotypes. 3. Cell cycle times were very similar (18-20 hr) when comparing the normal and cystic fibrosis lines or when comparing cystic fibrosis lines in phases 1 and 2 of growth. 4. No significant variations in population doubling times or growth fractions could be attributed to age or sex of the biopsy donor. 5. Variability in growth fractions and doubling times was minimal through the eighth subculture period but was very great in older cultures (tenth subculture). 6. Changes in growth fractions and doubling times appear to be due to the possibility of "aging" of human diploid fibroblasts in culture rather than to the presence or absence of genes for cystic fibrosis. 7. It is strongly indicated that differences in cell kinetic parameters cannot be used as the basis for differentiation between cells derived from normal or cystic fibrosis genotypes.     

Growth and surface properties of dispase dissociated human fibroblasts

The dissociation of human fibroblast cultures with the bacterial neutral protease (Dispase II) was monitored by viability and growth measurements and was compared to the effect of trypsin and EDTA. Cell suspensions with high viability and excellent growth were obtained after 10 min incubation at 37 °C in 4 U/ml dispase in 0.02% EDTA. A two to three-fold increase in mitotic index occurred in the cultures within 48 h after dispase dissociation. The initial rate of aggregation was comparable to that of trypsin or EDTA dissociated cells, but attachment to a substratum and agglutination by Wheat Germ Agglutinin were markedly enhanced. The results indicate that dispase-EDTA provides a valuable alternative to the enzymatic dissociation with trypsin. Moreover, it is an additional tool for the dissociation of cultured cells and for the study of the surface properties of single cells.     

Actomyosin contractility controls cell surface area of oligodendrocytes

Background  

To form myelin oligodendrocytes expand and wrap their plasma membrane multiple times around an axon. How is this expansion controlled?     

A quantitative study of growth variability of tumour cell clones in vitro

Abstract.   Objectives : In this study, we quantify growth variability of tumour cell clones from a human leukaemia cell line. Materials and methods : We have used microplate spectrophotometry to measure growth kinetics of hundreds of individual cell clones from the Molt3 cell line. Growth rate of each clonal population has been estimated by fitting experimental data with the logistic equation. Results : Growth rates were observed to vary between different clones. Up to six clones with growth rates above or below mean growth rate of the parent population were further cloned and growth rates of their offspring were measured. Distribution of growth rates of the subclones did not significantly differ from that of the parent population, thus suggesting that growth variability has an epigenetic origin. To explain observed distributions of clonal growth rates, we have developed a probabilistic model, assuming that fluctuation in the number of mitochondria through successive cell cycles is the leading cause of growth variability. For fitting purposes, we have estimated experimentally by flow cytometry the average maximum number of mitochondria in Molt3 cells. The model fits nicely observed distributions in growth rates; however, cells in which mitochondria were rendered non-functional (ρ⁰ cells) showed only 30% reduction in clonal growth variability with respect to normal cells. Conclusions : A tumour cell population is a dynamic ensemble of clones with highly variable growth rates. At least part of this variability is due to fluctuations in the initial number of mitochondria in daughter cells.     

Density and cell cycle dependence of cell surface proteins in hamster fibroblasts

The large, external, transformation-sensitive (LETS) glycoprotein of hamster fibroblasts, detected by lactoperoxidase-catalyzed iodination, is shown to vary in its accessibility at the cell surface, depending upon the growth state and position in the cell cycle. High levels correlate with arrest in early G1, and these fall after growth stimulation by serum. Very low levels are detectable on mitotic cells.     

A histochemical study in Huntington's disease and control cases.

1. Extracellular deposits of cerebrosides and free fatty acids were found in the formaldehyde fixed frozen sections of the frontal lobe in 8 cases of Huntington's disease, in one case of the infantile form of Gaucher's disease, 2 cases of Krabbe's globoid cell leucodystrophy, 2 cases of metachromatic leucodystrophy, one case with multiple sclerosis, 2 cases with cerebral contusion and one case with bacterial meningitis. 2. The cerebroside deposits were present in the white matter as well as in the grey matter. 3. The significance of these findings in relation to their etiology is discussed.     

Heme controls the expression of cell cycle regulators and cell growth in HeLa cells

Heme plays a central role in oxygen utilization and in the generation of cellular energy. Here we examined the effect of heme and heme deficiency on cell cycle progression and the expression of key regulators in HeLa cells. We found that inhibition of heme synthesis causes cell cycle arrest and induces the expression of molecular markers associated with senescence and apoptosis, such as increased formation of PML nuclear bodies. Our data show that succinyl acetone-induced heme deficiency increases the protein levels of the tumor suppressor gene product p53 and CDK inhibitor p21, and decreases the protein levels of Cdk4, Cdc2, and cyclin D2. Further, we found that heme deficiency diminishes the activation/phosphorylation of Raf, MEK1/2, and ERK1/2-components of the MAP kinase signaling pathway. Our results show that heme is a versatile molecule that can effectively control cell growth and survival by acting on multiple regulators.     

Impact of limiting visual input on gait: Individuals with Parkinson disease,age-matched controls,and healthy young participants

Normal and limited vision gait was investigated in individuals with Parkinson disease (PD), healthy older and healthy young individuals. Participants walked a GAITRite mat with normal vision or vision of lower limbs occluded. Results indicate individuals with PD walked more slowly, with shorter and wider steps, and spent more time in double support with limited vision as compared to full vision. Healthy young and old individuals took shorter steps but were otherwise unchanged between conditions.     

Mannose 6-phosphate/insulin-like growth factor II receptor in I-cell disease fibroblasts: increased synthesis and defective regulation of cell surface expression.

The amount of mannose 6-phosphate/IGF II receptors in fibroblasts from five I-cell patients was about 2-fold higher than in control fibroblasts. The elevated receptor concentration, which led to a higher binding and uptake of mannose 6-phosphate containing ligands and to a higher binding of IGF II resulted from an increased rate of synthesis, while the stability of the receptor was comparable to that in control fibroblasts. Control fibroblasts respond to mannose 6-phosphate, IGF I, IGF II and tumor promoting phorbol esters with a rapid redistribution of mannose 6-phosphate/IGF II receptors from internal membranes to the cell surface. In I-cell fibroblasts only a moderate increase in cell surface receptors was seen after exposure to these effectors. In contrast to control fibroblasts the treatment of I-cell fibroblasts with lysosomotropic amines failed to affect the mannose 6-phosphate containing ligand binding to the receptor. These data provide evidence for multiple potential regulatory sites in intracellular mannose 6-phosphate/IGF II receptor pathway which differ in control and I-cell fibroblasts.     

Comparison of actin and cell surface dynamics in motile fibroblasts

We have investigated the dynamic behavior of actin in fibroblast lamellipodia using photoactivation of fluorescence. Activated regions of caged resorufin (CR)-labeled actin in lamellipodia of IMR 90 and MC7 3T3 fibroblasts were observed to move centripetally over time. Thus in these cells, actin filaments move centripetally relative to the substrate. Rates were characteristic for each cell type; 0.66 +/- 0.27 microns/min in IMR 90 and 0.36 +/- 0.16 microns/min in MC7 3T3 cells. In neither case was there any correlation between the rate of actin movement and the rate of lamellipodial protrusion. The half-life of the activated CR-actin filaments was approximately 1 min in IMR 90 lamellipodia, and approximately 3 min in MC7 3T3 lamellipodia. Thus continuous filament turnover accompanies centripetal movement. In both cell types, the length of time required for a section of the actin meshwork to traverse the lamellipodium was several times longer than the filament half-life. The dynamic behavior of the dorsal surface of the cell was also observed by tracking lectin-coated beads on the surface and phase-dense features within lamellipodia of MC7 3T3 cells. The movement of these dorsal features occurred at rates approximately three times faster than the rate of movement of the underlying bulk actin cytoskeleton, even when measured in the same individual cells. Thus the transport of these dorsal features must occur by some mechanism other than simple attachment to the moving bulk actin cytoskeleton.     

Qualitative and quantitative characteristics of the extracellular DNA delivered to the nucleus of a living cell

Background  

The blood plasma and other intertissue fluids usually contain a certain amount of DNA, getting there due to a natural cell death in the organism. Cells of this organism can capture the extracellular DNA, whereupon it is delivered to various cell compartments. It is hypothesized that the extracellular DNA is involved in the transfer of genetic information and its fixation in the genome of recipient cell.