GD3 ganglioside is prevalent in fully differentiated neurons from rat retina

Adult mammalian retinas contain unusually high amounts of GD3, a ganglioside of the lactosylceramide series. In this respect, they differ from adult avian retina and other regions of the adult avian and mammalian brain, where GD3 is a minor ganglioside and gangliosides of the gangliotetraosylceramide series (GM1, GD1a, GD1b, GT1b) are the predominant ones. We compare here the ganglioside patterns of rat, human, horse, and guinea pig retinas, which are known to differ in the degree of vascularization and astrocytic cell content. All these retinas showed a prevalence of pathway "b" gangliosides over pathway "a" gangliosides but showed no correlation between GD3 content and the degree of vascularization and astrocytic cell content. Immunostaining of rat retina sections showed the presence of GD3 in the inner and outer plexiform layers and also in the ganglion cell and inner nuclear layers. About 60% of the cells dissociated from rat retina showed immuno-colocalization of GD3 and the neuronal marker class III beta tubulin isotype or cholera toxin binding. All morphologically identifiable glial Muller cells coexpress GD3 and gangliotetraosylgangliosides. GD3 was a minor ganglioside among these axonally transported by ganglion cells in rats and guinea pigs, suggesting that it is either not synthesized by ganglion cells or, if so, it is restricted to the cell soma and/or dendritic tree. Our results demonstrate that, unlike neurons from avian retina and other regions of avian and mammalian brain, neurons from mammalian retina not only contain gangliosides of the gangliotetraosylceramide series but also keep a prevalence of gangliosides of the lactosylceramide series (GD3) when they are fully differentiated.     

Multiple sclerosis is associated with enhanced B cell responses to the ganglioside GD1a

The occurrence and role of autoantibodies to gangliosides and other lipid-containing components of the central nervous system in Multiple Sclerosis (MS) are unsettled. Using sensitive ELISAs, we measured IgG and IgM antibody titers and absorbances to the three major gangliosides GD1a, GD1b and GM1, and to sulfatides, cardiolipin and myelin proteins in paired serum and cerebrospinal fluid (CSF) from patients with untreated MS, optic neuritis (ON), acute aseptic meningo-encephalitis (AM) and other neurological diseases (OND). Twenty-three per cent of 30 MS (P<0.04) and 18% of 32 ON patients (P<0.05) presented elevated IgG antibody titers to GD1a in serum compared to 9% of patients with OND. Six (40%) of the patients with malignant MS had elevated serum IgG antibody titers to GD1a compared to one (6%) of the patients with benign MS (P<0.04). In CSF, elevated IgG antibody titers to GD1a were measured in 13% of MS and 20% of ON patients compared to 4% of patients with OND (P<0. 03 and P<0.02, respectively). The augmented IgG response to GD1a in serum also separated MS from Guillain-Barré syndrome. Compared to OND increased IgM absorbances to sulfatides and cardiolipin were observed in CSF of patients with MS, but also in AM. Elevated IgG antibody titers to myelin proteins were found more often in MS patients' serum and MS, ON and AM patients' CSF compared to OND. The data implicate that among the multitude of enhanced B-cell responses occurring in MS and ON, that directed to GD1a is common and more discriminative, and should be evaluated in future MS treatment studies.     

Expression and function of ganglioside 9-O-acetyl GD3 in postmitotic granule cell development

We have shown previously that the Jones monoclonal antibody (Jones mAb) recognizes 9-O-acetyl GD3 expressed during periods of neuronal migration and neurite outgrowth in the developing rat nervous system. In the present study we investigated the expression of this ganglioside in the developing cerebellum and correlated this expression with granule cell migration. Electron microscopic immunocytochemistry revealed that around the peak of cerebellar neuronal migration (7-day-old rat), 9-O-acetyl GD3 was localized at the contact sites between migrating granule cells and radial glia in the external granular layer and prospective molecular layer. In addition, using microexplant and slice cultures of the postnatal rat cerebellum, we tested whether the ganglioside detected by our antibody contribute to the regulation of neuronal migration in the cerebellar cortex. We have shown that the Jones mAb blocks the migration of neurons in a dose-dependent manner. These findings suggest strongly that 9-O-acetyl GD3 is involved in granule cell migration in the developing cerebellum.     

A reappraisal of ganglioside GD3 expression in the CNS

G_D3 ganglioside is a major glycolipid component of the developing central nervous system but diminishes considerably as the CNS matures. Despite consistent biochemical data, the cellular localization of G_D3 expression has been controversial. In this commentary we will review the cellular expression of G_D3 during CNS development and in neuropathological circumstances as determined by studies with the two most commonly used anti G_D3 monoclonal antibodies, R24 and LB1. G_D3 is not restricted to any one cell lineage, being expressed in development to varying degrees by immature neuroectodermal cells, oligodendrocyte progenitors, ameboid microglia, and subpopulations of developing neurons and astrocytes. In the adult CNS, G_D3 is expressed in low amounts by some neuronal subpopulations, on reactive and resting microglia, and by reactive astrocytes. In the appropriate contexts of development or neuropathology, anti-G_D3 antibodies are useful for cell type identification and for cell isolation, but caution should be exercised because of the lack of cellular specificity. © 1996 Wiley-Liss, Inc.     

Demonstration of ganglioside GD3 in human reactive astrocytes

Astrocytes are the cells that actively participate in the process of lesion repair in the central nervous system (CNS), and reactive astrocytosis of varying degrees becomes apparent with time in any pathological condition occurring in the normally developed postnatal CNS. Ganglioside GD3 (II3a(NeuAca2-8NeuAc)-LacCer, GD3) in reactive astrocytes from autopsied patients with Creutzfeldt-Jakob disease (CJD) and old cerebral infarction was investigated immunocytochemically, using mouse IgM anti-GD3 monoclonal antibody (DSG-1). Reactive astrocytes in CJD and cerebral infarction demonstrated GD3-immunoreactivity within the cytoplasm. Normal astrocytes were negative. The present data raise the possibility that GD3 in reactive astrocytes has biological implications for the properties of the cells, such as cellular motility.     

Immunostaining of ganglioside GD1b, GD3 and GM1 in rat cerebellum: cellular layer and cell type specific associations

We have studied the cellular distribution of gangliosides GD1b, GD3 and GM1 in rat cerebellum by immunostaining, using monoclonal antibodies and confocal microscopy. Antibodies against astroglial, neuronal and synaptic vesicle associated molecules were used for colocalization analyses. In the gray matter, the anti-GD1b antibody stained thin strands in the molecular layer (ML), interpreted as Bergman glia fibers based on colocalized staining with anti-glial fibrillary acidic protein (GFAP). The neuropil in the granule (GL) and Purkinje (PL) cell layers was also anti-GD1b positive. The anti-GD3 antibody stained the ML, the neuropil in the GL and PL and also the granule and Purkinje cell bodies, appearing intracytoplasmically and vesicle associated. Anti-GD1b and anti-GD3 staining in the GL glomeruli were colocalized with anti-synaptophysin staining. The anti-GM1 antibody stained cell bodies in the ML but they could not be characterized in colocalization experiments. The GL and PL were not stained with the anti-GM1 antibody. In the white matter, different staining patterns were seen for the gangliosides, the anti-GM1 staining being the most intense. This study shows cellular layer and cell type specific associations of the investigated gangliosides and localization of GD1b and GD3 at synaptic sites, warranting further studies on their role in synaptic mechanisms.     

A gradient molecule in developing rat retina: expression of 9-O-acetyl GD3 in relation to cell type, developmental age, and GD3 ganglioside

In the embryonic and postnatal rat retina a cell surface antigen that is detected by monoclonal antibody JONES is distributed in a dorsoventral gradient. Biochemical analysis has determined that the antigen is a modified ganglioside, 9-O-acetyl GD3. In the present study, the distributions of 9-O-acetyl GD3 and its possible precursor GD3 in developing rat retina were compared immunocytochemically using specific monoclonal antibodies JONES and R24. On embryonic day 13 (E13) immunoreactivity to JONES was localized to central retina; however, R24 stained throughout the retinal epithelium. By E20, when JONES binding was distributed in a gradient along the dorsoventral axis, R24 again stained dorsal and ventral regions with uniform intensity. Analysis of freshly dissociated retinal cells further revealed that GD3 and 9-O-acetyl GD3 expressions do not necessarily coincide. At E15 and postnatal day 2 (PN2), the majority of cells (78 and 92%, respectively) were immunolabeled by antibody to GD3, while between E15 and PN2 the percentage of cells immunolabeled by antibody to 9-O-acetyl GD3 rose from 19 to 68%. By PN4, labeling decreased for both molecules; however, the rate of decline in 9-O-acetyl GD3 labeling was more pronounced. Regulation of the numbers of JONES-positive cells does not appear to depend on interaction with the extraretinal environment, for in neural retina explanted at E15 the proportions of 9-O-acetyl GD3-bearing cells was found to be similar to the percentage observed in neural retina developing to an equivalent age in vivo. Experiments to identify the retinal cell types bearing 9-O-acetyl GD3 revealed that it is expressed by both neurons and glia growing in monolayer cultures of rat perinatal neural retina. Examination of freshly dissociated retinal cells following simultaneous labeling for some specific cell types and 9-O-acetyl GD3 demonstrated that the latter determinant is present on photoreceptor, amacrine, and ganglion cells. For each neuronal cell type, however, not all of the cells were immunoreactive with JONES. We conclude that the differences in the percentages of JONES- and R24-positive cells, and in particular the different rates at which JONES and R24 staining declined with age, indicate that the expression of the JONES epitope is regulated with some independence from parent ganglioside GD3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of GD3 ganglioside by developing rat cerebellar Purkinje cells in situ

GD3 is a major ganglioside of the immature vertebrate CNS, and its expression is suggested to be characteristic of immature neuroectodermal cells. Using immunocytochemistry on cryostat sections of developing rat cerebellum with a monoclonal antibody specific for GD3, we have found that GD3 begins to be expressed on the plasma membrane of Purkinje cell bodies and dendrites beginning at postnatal day 7. Staining became brighter as the dendritic tree of the cells enlarged. As the Purkinje cells began to mature in different folia, they became GD3+, until by 15 days postnatal all Purkinje cells were GD3+. Positive staining of the dendritic tree was still present in the adult cerebellum. Using a monoclonal antibody 7-8D2, which recognizes cerebellar granule cells and their axons (the parallel fibres), and polyclonal antibodies against a synaptic vesicle component synaptophysin, double-immunofluorescence staining together with anti-GD3 antibodies suggested that the appearance of GD3 immunoreactivity did not correlate either with the ingrowth of parallel fibres or the presence of their synapses on Purkinje cell dendrites. However, comparison with earlier morphological studies showed that the appearance of GD3 immunoreactivity correlated well with the formation of climbing fibre synapses on Purkinje cell dendrites and the onset of the rapid expansion of the dendritic tree. These results are in keeping with the idea that elevated GD3 concentrations are found in certain cell types during periods of rapid growth or high metabolic activity but also show that this is not only restricted to immature cells.     

星形胶质细胞和神经元凋亡的细胞周期时相分布特征

目的 通过测定星形胶质细胞和神经元各自细胞凋亡的细胞周期特异性分布规律,以进一步探讨两种细胞不同的凋亡发生机制. 方法 建立大脑中动脉阻塞再灌注模型并取损伤后3 d的脑组织进行分析,缺血再灌注3 d后取脑,采用BrdU方法标记S期的细胞;再利用流式细胞分选技术特异性的将星形胶质细胞和神经元分选出来,采用BrdU标记并结合TUNEL方法,分析星形胶质细胞和神经元凋亡的细胞周期定位. 结果 与正常组比较,缺血再灌注组中缺血侧大脑皮层可见BrdU标记阳性细胞和TUNEL标记阳性细胞均明显增多,并可见TUNEL阳性和BrdU阳性双重标记的细胞.星形胶质细胞凋亡可以发生在细胞周期的各个时相,大多发生在G0/G1期,比神经元在G0/G1期的细胞凋亡率显著增高(P＜0.01);神经元进入S期的细胞凋亡率显著高于星形胶质细胞(P＜0.01). 结论 星形胶质细胞和神经元的细胞凋亡是多点启动的,脑缺血后两种细胞的凋亡具有不一致的细胞周期时相性分布特征.     

Kainic acid-induced apoptosis in cerebellar granule neurons: an attempt at cell cycle re-entry

This study was undertaken to investigate whether kainic acid (KA) may regulate the expression of several proteins which plays an important role in cell-cycle progression in cerebellar granule neurons (CGNs). KA induced decrease in MTT values in a concentration dependent way. Flow cytometric analysis showed that KA was able to induce 30% apoptosis in CGNs. Apoptotic nuclear condensation were detected 24 h of exposure to KA (200 microM). An associated marked increase in DNA synthesis, measured by BrdU incorporation, was observed. Western blot analysis showed that KA induced an increase in the expression of Cdk2, cyclin E and E2F-1. It is proposed that, in post-mitotic cells like CGNs, re-entry cell cycle could be responsible for the apoptotic effect of KA.     

Restricted occurrence of an unusual ganglioside GD1α in rat brain and its possible involvement in dendritic growth of cerebellar Purkinje neurons

The spatial pattern of expression of a minor disialosyl ganglioside GD1α in the rat brain was investigated immunohistochemically using a specific murine monoclonal antibody KA-17. The antibody shows noticeable immunoreactivity in the proximal dendrites and neuronal cell bodies of restricted populations of neurons including cerebral pyramidal neurons and cerebellar Purkinje neurons. Immunocytochemical analysis revealed that Purkinje neurons maintained in a dissociate culture condition also express GD1α in the dendrites and cell bodies. We have examined the functional involvement of this ganglioside in the growth of brain neurons using KA-17 antibody. Addition of the antibody to cerebellar primary cultures caused perturbation of the dendritic development of Purkinje neurons in a dose-dependent manner. The length and branching of the dendrites were severely decreased by the antibody treatment. When other anti-glycoconjugate or sphingolipid monoclonal antibodies were tested, only HNK-1 monoclonal antibody that recognizes sulfoglucuronic residues in glycolipids and glycoproteins had similar but moderate inhibitory actions on the dendritic development of these neurons. In contrast to the morphological alterations observed in Purkinje cells, other cerebellar cells including granule neurons appear to be almost normal following the antibody treatment. These observations lead to the possibility that GD1α ganglioside has a role in the development of Purkinje cell dendrites. © 1996 Wiley-Liss, Inc.     

Ganglioside GD3 of cerebral neurons and Purkinje cells in aged human brains

Immunocytochemical staining for ganglioside GD3 (II^3α (NeuAcα2–8NeuAc)-LacCer, GD3) in neuronal cells of the cerebral cortices (cerebral neurons), and cerebellar dentate nucleus (dentate neurons) and Purkinje cells, in human autopsy cases of progressive supranuclear palsy, senile dementia of the Alzheimer type, Pick's disease, amyotrophic lateral sclerosis and olivopontocerebellar atrophy (OPCA) was undertaken using mouse IgM anti-GD3 monoclonal antibody. Cerebral neurons and dentate neurons were constantly GD3-immunoreactive and immunoreactivity was observed in the cytoplasm. The peroxidase reaction product for GD3 (RP) in cerebral and dentate neurons was granular in appearance. It appeared that RP was associated with lipofuscin granules. However, immunoreactivity of Purkinje cells varied among cases, and the RP was slightly granular even when they were positive. This study suggests that lipofuscin granules contributed to the neuronal immunoreactivity of GD3 in aged human brains.     

Antibodies to the ganglioside GD1b in a patient with motor neuron disease and thyroid adenoma

Patients with motor neuron disease with thyroid disorders have been described, although the relationship between the two conditions is unclear. We treated a patient with amyotrophic lateral sclerosis who also had a follicular adenoma of the thyroid gland. Because thyroid gland plasma membranes contain high concentrations of complex gangliosides, such as GD1b, and some patients with motor neuron disease have IgM antibodies to GD1b, we decided to assay serum from this patient for the presence of antiganglioside antibodies. IgM antibodies to GD1b were detectable at serum dilutions of 1:500 and 1:1000 by enzyme-linked immunosorbent assay. While these titers are less than those usually described in patients with plasma cell dyscrasia, they are well in excess of normal values. Antibody to GM1 was also detectable at a lower (1:100) dilution. We do not know the importance of the anti-GD1b antibodies in this patient, but it is possible that antibodies to GD1b are involved in this and other cases of motor neuron disease associated with thyroid disease.     

GOLPH3对人脑胶质瘤细胞周期及凋亡调节作用的研究

目的探讨GOLPH3(Golgi phosphoprotein 3)对人脑胶质瘤细胞周期及凋亡的影响及调控机制。方法在U251胶质瘤细胞中利用siRNA下调GOLPH3的表达后,流式细胞术检测细胞周期和凋亡的变化,RT-PCR和Western blot技术检测细胞周期相关蛋白cyclin D1,p21~(waf1/cip)(p21)和p53在核酸和蛋白水平的变化,同时检测下调GOLPH3对Akt、p Akt蛋白水平的影响。结果 3条GOLPH3 siRNA均能下调GOLPH3的蛋白表达(均P0.05);下调GOLPH3使细胞周期阻滞在G0/G1期(P0.05),细胞凋亡率明显增加(P0.05)。同时发现下调GOLPH3使cyclin D1的核酸和蛋白水平降低,p21的核酸和蛋白水平升高而p53的核酸和蛋白水平无明显变化。下调GOLPH3的表达降低p Akt的蛋白水平(P0.05),但对Akt无影响。结论在U251细胞中下调GOLPH3使细胞阻滞在G0/G1期,促进细胞凋亡。下调GOLPH3对细胞周期的调节作用可能是通过抑制PI3K-Akt信号通路,进而下调cyclin D1和上调p21的表达来实现的。     

Amoeboid microglia expressing GD3 ganglioside are concentrated in regions of oligodendrogenesis during development of the rat corpus callosum

In a recent study we demonstrated expression of the platelet-derived growth factor α receptor (PDGFRα) in cells of the early oligodendrocyte lineage that were identified as either G_D3 ganglioside + oligodendrocyte progenitors or O4 sulfatide + preoligodendrocytes. We also identified a subpopulation of G_D3 immunoreactive cells that did not express mRNA for the PDGF receptor. The distinct large amoeboid morphology of these cells was characteristic of cells in the macrophage lineage rather than in the oligodendrocyte lineage. To determine if the G_D3-positive but PDGFRα mRNA-negative cells were in the macrophage lineage, we compared the spatial and temporal expression patterns of G_D3 ganglioside and ED1, a macrophage-specific antigen. Analysis prenatally indicated that at embryonic day 15, ED1+ and G_D3 + cell populations resided in the subpial connective tissue. At embryonic day 21, these two populations were seen in a region extending from the lateral ventricle through the subventricular and intermediate zones. In this study we report that these large, round, G_D3 immunoreactive cells have the same cell morphology and anatomical distribution as the ED1 immunoreactive cells. Both cell populations contained pyknotic nuclei within their cytoplasm. Furthermore, the G_D3+/PDGFRα- cells appear to be involved in clearing cellular debris in regions of gliogenesis. These data suggest that this subpopulation of G_D3 immunoreactive cells belongs to the microglia/macrophage lineage. © 1995 Wiley-Liss, Inc.     

Inhibition of neuronal migration by JONES antibody is independent of 9-O-acetyl GD3 in GD3-synthase knockout mice

It has been shown previously that the migration of granule neurons in neonatal cerebellum can be inhibited by a monoclonal antibody (Mab) JONES. Because the inhibition is presumed to be mediated through binding of the JONES antibody to 9-O-acetyl GD3, we used GD3-synthase knockout (GD3S-/-) mice that do not express 9-O-acetyl GD3 and also have no detectable defect in brain development, to examine the mechanism of the inhibitory effect. We found no difference between the migration of granule neurons in the neonatal cerebellar explant culture in GD3S-/- mice and in wild-type mice. Addition of the Mab JONES, but not Mab R24 or A2B5, in the culture medium blocked the neuronal migration in the explant culture of the wild-type mice. The inhibitory effect of Mab JONES was also observed, however, in the explant culture of GD3S-/- mice. Immuno-HPTLC analysis showed at least two JONES-positive glycolipids bands in the lipid extract of GD3S+/+ mice, and none was detected in that of GD3S-/- mice. Western blot analysis of the cerebellum homogenate of wild-type and GD3S-/- mice identified at least 3 JONES-positive protein bands, one of which is beta1-integrin. Because the JONES antibody also blocked neuronal migration in the cerebellar explant culture of GD3S-/- mice that do not express 9-O-acetyl-GD3, it suggested an alternative mechanism for the inhibitory effect of the antibody, at least in the GD3S knockout mice, and the inhibitory effect of the JONES antibody on neuronal migration could be mediated through its binding to beta1-integrin.     

Glycolipid sialosyltransferase activity in synaptosomes exhibits a product specificity for (2-8)disialosyl lactosyl ceramide (ganglioside GD3)

Intact synaptosomes prepared from 28-day-old rat brains were incubated with CMP-N-acetyl-(14C) neuraminic acid in Krebs-Henseleit buffer in an atmosphere of 95% O2: 5% CO2, at 37 degrees C. The activity of CMP-NANA:ganglioside sialosyltransferase using endogenous acceptors was 0.84 pmoles NANA transferred/mg synaptosomal protein/hr. Analysis of the distribution of labeled sialic acid revealed that GD3 ganglioside (alpha 2----8 disialosyl, alpha 2----3 galactosyl, beta 1----4 glucosyl, beta 1----1-ceramide) was the major product in the membrane carrying 32% of the total lipid bound label. Treatment of the reaction products with Clostridium neuraminidase liberated labeled sialic acid from GD3 and yielded labeled GM3, then unlabeled lactosyl ceramide. Lac-cer and GM3 are present in small amounts in synaptosomes, and GD3 represents less than 2% of the total ganglioside. Our findings indicate that the sialosyltransferase activity of synaptosomes exhibits a preferential product specificity for the small pool of synaptosomal membrane GD3 ganglioside that may be formed in situ, via sialosylation of its precursor (GM3 or lactosyl ceramide) which pre-exists in the synaptosomal plasma membrane. The second major labeled product quantitatively was GD1a whose precursor substrate, GM1, is quite abundant in the membrane, so that the conversion rate of GM1 to GD1a was low in comparison with GD3 formation. Sialosylation of other synaptosomal membrane gangliosides was negligible.     

Receptor for the C3a anaphylatoxin is expressed by neurons and glial cells

Little is known about the expression of the receptor for complement anaphylatoxin C3a (C3aR) in the central nervous system (CNS). In this study, we provide the first evidence that neurons are the predominant cell type expressing C3aR in the normal CNS. By using in situ hybridization (ISH) and immunohistochemistry, we found that C3aR is constitutively expressed at high levels in cortical and hippocampal neurons as well as in Purkinje cells. Moreover, we showed that primary culture of human astrocytes and microglia express the C3aR mRNA as assessed by RT‐PCR. In situ hybridization performed on rat primary astrocytes confirmed the RT‐PCR result demonstrating C3aR expression by astrocytes. In experimental allergic encephalitis (EAE), C3aR expression was elevated on microglia, infiltrating monocyte‐macrophage cells and a few astrocytes, whereas neuronal expression remained unchanged during the course of the disease. These data demonstrate that the C3aR is expressed primarily by neurons in the normal CNS and that its neuronal expression is not dramatically upregulated under inflammation. This is in contrast to the increased neuronal expression of the C5aR in several inflammatory CNS conditions. The high constitutive expression of the C3aR by neurons suggests this receptor may play an important role in normal physiological conditions in the CNS. GLIA 26:201–211, 1999. © 1999 Wiley‐Liss, Inc.     

Selective cell-cycle arrest and induction of apoptosis in proliferating neural cells by ganglioside GM3

Control of cell proliferation and cell survival is critical during development of the vertebrate central nervous system (CNS). Much of the cell death seen during early stages of CNS development occurs through apoptosis; however, the factors that induce this early apoptosis are not clearly understood. Gangliosides, sialylated glycosphingolipids, are expressed in the CNS and have been proposed to regulate cell growth and differentiation. Here we show that the simple ganglioside GM3 selectively inhibits the proliferation of and induces apoptosis of actively dividing astrocyte precursors and other neural progenitors. The inhibition of astrocyte precursor proliferation by GM3 appears to be mediated in part by the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1). During neonatal development there is extensive cell proliferation and little apoptosis in the ventricular and subventricular zones of the CNS. This proliferation was dramatically inhibited and the degree of apoptosis dramatically increased following intraventricular administration of GM3. These data suggest that GM3, a simple ganglioside, may regulate cell proliferation and death in the CNS and as such may have potential for brain tumor therapy.     

S-100 protein synthesis in cultured glioma cell is G1-phase of cell cycle dependent

The kinetics of S-100 protein synthesis was studied in synchronized cultured glioma (C₆) cells. The peak of the synthesis was detected at G₁-phase of the cell cycle and apparently was not population density-dependent, since sparsely cultured G₁-phase cells synthesized the same proportion of the protein per cell as that of confluent cultures, whereas the total cellular protein synthesis was found steadily increasing throughout the intermitotic phases.