αγ-Enolase in the Rat: Ontogeny and Tissue Distribution

Abstract: The rat brain enolases are dimers composed of α and γ subunits. At pH 8.6 αγ-enolase seemed to be stable, and no evidence was found for the possible formation of αγ-enolase from αα-enolase and γγ-enolase in the course of rat brain homogenization. During ontogeny of the rat forebrain, αγ-enolase was formed before γγ-enolase. The half-maximal specific concentrations were reached at postnatal days 14 and 23, respectively. The distribution of αγ- and γγ-enolase in various rat brain areas was also investigated. In all areas both forms were present. In neuroendocrine tissues αγ-enolase was present at a much higher concentration than γγ-enolase. The ratio between γγ-enolase and αγ-enolase may be indicative of the degree of neuronal maturation, a conclusion further substantiated by the high ratio observed in cerebellum and the low ratio observed in olfactory bulbs, both compared with the ratio in forebrain.     

Enolase Activity and Isoenzyme Distribution in Human Brain Regions and Tumors

Abstract: The distribution of enolase (EC 4.2.1.11) activity and isoenzymes in various regions of human brain at different ages (from 23 weeks of gestation to 95 years) and in brain tumors has been determined. Total enolase activity increased in all regions with age. No significant differences were found in the relative proportions of αα-, αγ-, and γγ-enolase isoenzymes in the various brain regions, determined by agarose gel electrophoresis. Type αα-enolase was the predominant isoenzyme, and αγ-enolase represented a substantial proportion of the total enolase activity. Astrocytomas, anaplastic astrocytomas, glioblastomas, and meningiomas possessed lower enolase activity than normal brain. Among astrocytic tumors, total enolase activity correlated with malignancy. Astrocytomas possessed the lowest and glioblastomas the highest enolase activity. All tumors possessed a higher proportion of αα-enolase and a lower proportion of γγ-enolase than the normal human brain. Among astrocytic tumors, glioblastomas were the tumors with the highest proportion of αα-enolase and lowest proportion of γγ-enolase.     

Plasminogen Binds Specifically to α-Enolase on Rat Neuronal Plasma Membrane

Abstract: Plasminogen (PGn) that we identified in microglial-conditioned medium has a neurotrophic factor-like effect on cultured neurons. We have also shown that PGn binds specifically to a protein with a molecular mass of 45 kDa in the neuronal plasma membrane. As a candidate PGn receptor-like molecule on the neuronal surface, this 45-kDa protein was purified from the plasma membrane of embryonic rat brain. Amino acid sequence analysis of polypeptides derived from the cleavage of the protein with cyanogen bromide and V8 protease revealed that the 45-kDa protein is identical to rat α-enolase. In fact, PGn was found to bind to purified rat α-enolase and also to a synthetic peptide (30 residues) that corresponds to the carboxyl terminal region of rat α-enolase. Physical properties of the 45-kDa protein, such as molecular mass, isoelectric point, and the ability to form dimers, are quite similar to those of α-enolase. The 45-kDa PGn-binding protein in the plasma membrane was also recognized by anti-rat α-enolase antibody, and pretreatment with α-enolase antibody markedly diminished the PGn-binding to the plasma membrane. In addition, immunocytochemical staining of the cultured cells under the nonpermeable condition showed that α-enolase is present on the cell surface of a certain population of neurons. These results suggest that α-enolase may function as a PGn-binding molecule on the neuronal cell surface.     

Human γγ-Enolase: Two-Site Immunoradiometric Assay with a Single Monoclonal Antibody

Abstract: A monoclonal antibody (mAb), termed BBS/NC/VI-H14 (H14), that reacts with the human enzyme γγ-enolase was prepared. It was directed against the γ-subunit and did not cross-react with the α- or β-subunit. The mAb H14 can be used for quantitative determination of γγ-enolase in a two-site immunoradiometric assay (two-site IRMA). It is also suitable for immunostaining formalin-fixed tissues. The specific identification of γγ-enolase provided by the two-site IRMA with H14 is discussed in relation to the cellular distribution of this protein.     

DIFFERENTIATION AND DEDIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN SHORT- AND LONG-TERM CULTURES

The crystallin synthesis of rat lens cells in cell culture systems was studied in relevance to their terminal differentiation into lens fibers. SDS-gel electrophoresis combined with several immunological techniques showed that γ-crystallin is a fiber-specific lens protein and is not localized in the epithelium of either newborn or adult lenses. When lens epithelial cells of newborn rats were cultured in vitro , α-crystaIlin was detected in many, but not all, of cells cultured for 10 days. Cells with α-crystallin gradually changed their shape into a flattened filmy form and finally differentiated into lentoid bodies. The differentiation of lentoid bodies was also found in cultures of epithelial cells obtained from adult lenses. The molecular constitution of lentoid bodies was the same as that of lens fibers in situ . The differentiation of lentoid bodies occurred successively for 5 months in cultures of lens epithelial cells. Most of the proliferating cells, however, lost α-crystallin during the culture period. Thereafter, they did not show any sign of further differentiation into lens fibers. Four clonal lines were established from these cells. One protein which is specific to the lens epithelium and the neural retina in situ (tentatively named as β_u-crystallin) was maintained in all lines, suggesting that some specific properties of ocular cells remain in the lined cells.     

Channel expression correlates with differentiation stage during the development of oligodendrocytes from their precursor cells in culture

Membrane currents in cultured murine oligodendrocytes and their precursors were characterized using the patch-clamp technique. Prior to recording, cells were identified by immunofluorescence using monoclonal antibodies characteristic of two types of precursor cells and two differentiation stages of oligodendrocytes. The most immature, A2B5 antigen-positive glial precursors, expressed four types of voltage-activated K+ currents and tetrodotoxin-sensitive Na+ currents. The more differentiated cells, O4 antigen-positive glial precursors, expressed similar K+ currents, but Na+ currents were recorded in only a minority of cells. In differentiated O1 and O10 antigen-positive oligodendrocytes the channels characteristic of precursor cells were no longer observed, but an inwardly rectifying K+ current was apparent. Thus, channel expression by cells of the oligodendrocyte lineage correlates with differentiation stage and is more complex in precursor cells than in oligodendrocytes.     

Involvement of tazarotene-induced gene 1 in proliferation and differentiation of human adipose tissue-derived mesenchymal stem cells

Objective:  Mesenchymal stem cells (MSC) have both self-renewal and multilineage differentiation potential, and bone marrow-derived MSC have been applied for tissue regeneration and repair. Although adipose tissue-derived MSC (ASC) have emerged as an alternative cell source, little information is available regarding the biologic difference between ASC derived from visceral and subcutaneous fat. Therefore, we aimed to compare the proliferation and gene expression profile of cultured human visceral ASC (VASC) and subcutaneous ASC (SASC), and to identify a novel gene involved in proliferation and differentiation of ASC.
Materials and methods:  We performed microarray analysis of cultured VASC and SASC, and investigated the role of tazarotene-induced gene 1 (TIG1), a most differentially expressed gene, in the proliferation and differentiation of ASC.
Results:  SASC proliferated faster than VASC for over 10 passages, and TIG1 expression was consistently up-regulated in VASC of humans, rats and mice. Overexpression of the TIG1 gene in human SASC inhibited cell proliferation, whereas knockdown of TIG1 expression by siRNA promoted cell proliferation. In addition, overexpression of the TIG1 gene in SASC enhanced their differentiation into adipocytes, and promoted up-regulation of peroxisome proliferators-activated receptor γ and CCAAT/enhancer binding protein α. On the other hand, TIG1 overexpression in SASC inhibited their differentiation into osteocytes and the expression of osteocalcin.
Conclusion:  TIG1 plays an important role in regulating proliferation and differentiation of ASC.     

Changes in the Expression of the αα Form of Enolase During Neuroblastoma Differentiation

Abstract: The relative amounts of the different enolase isozymes present in neuroblastoma cells change during differentiation. When differentiation is induced by low serum in the presence of DMSO (dimethyl sulfoxide), there is a 50% decrease in the concentration of enolase activity associated with the form αα, and an increase in the activity associated with the γ-containing isozymes (αγ plus γγ); in the absence of DMSO, there is no decrease in αα or in total enolase activity. In order to study the mechanism of the changes in αα, cells differentiated with low serum with and without DMSO were compared. Measurements of the concentration of the α antigen by microcomplement fixation and by immunotitration demonstrate that the decreased enolase activity in DMSO cells is due to a decreased concentration of the α antigen. Measurements of the relative rate of synthesis of the antigen show that the decreased concentration of the α antigen is due to a decreased rate of synthesis. Enolase in differentiated cells is sufficiently stable (t_1/2 > 100 h) that a comparison of the relative rates of degradation has not been possible. The decreased synthesis of the α subunit of enolase that occurs under these conditions appears to be a useful model system for studying the de-expression of the α gene that occurs in vivo during neuronal differentiation.     

Telomerase and oligodendrocyte differentiation

Myelin in the mammalian central nervous system (CNS) is produced by oligodendrocytes, most of which arise from oligodendrocyte precursor cells (OPCs) during late embryonic and early postnatal development. Both external and internal cues have been implicated in regulating OPC exit from the cell cycle and differentiation into oligodendrocytes. In this study, we demonstrate that differentiation of cultured OPCs into mature oligodendrocytes is associated with lower levels of activity of telomerase, the ribonucleoprotein that synthesizes telomeric DNA at the ends of chromosomes. Differentiation is also associated with lower levels of mRNA encoding the catalytic subunit of telomerase (TERT), whereas no difference is seen in the expression of its telomeric template RNA component (TR). These data suggest a possible role for telomerase during normal growth and differentiation of oligodendrocytes that may be relevant to the mechanism of myelination in the CNS. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 224–234, 2001     

Blood Platelets Contain a Neuron-Specific Enolase Subunit

Abstract: Neuron-specific enolase (NSE) is a cell-specific isoenzyme of the glycolytic enzyme enolase that is present only in neurons and selected neuroendocrine cells. We now report the presence of this neuronal marker in blood platelets. The level of NSE found in human blood platelets is much lower than that found in brain tissue (0.045% of the total soluble protein for platelets versus 1.5% for cortical tissue), but is 20-30 times higher than NSE levels found in peripheral non-nervous tissues. Chromatographic analysis indicates that the majority of the NSE γ-subunit in platelets is present as the hybrid αγ isoenzyme. This, coupled with the high level of non-neuronal enolase (NNE) found in platelets, indicates that blood platelets contain both the α- and γ- subunit.     

Increased Nervous System-Specific Enolases in Rat Plasma and Cerebrospinal Fluid in Bilirubin Encephalopathy Detected by an Enzyme Immunoassay

Abstract: Three forms of enolase isozymes (αα, αγ, and γγ), including nervous system-specific forms, were measured in the cerebrospinal fluid and the blood plasma of jaundiced or nonjaundiced infant rats by means of enzyme immunoassay systems capable of detecting each form of enolase at the 1 amol (10⁻¹⁸ mol) level. Average enolase levels in cerebrospinal fluid in normal rat were 2.0, 0.2 and 0.1 pmol/ml for αα, αγ, and γγ forms, respectively. Levels of αγ and γγ forms (nervous system-specific enolases; NSE) in jaundiced rats, which suffer Purkinje cell degeneration due to the inborn hyperbilirubinemia, were three to four times as high as the normal values. When kernicterus was induced in jaundiced rats by an injection of bucolome, the NSE level in cerebrospinal fluid was elevated up to more than 30-fold the control, together with a significantly higher level of αγ form in blood plasma. These results suggest that assays of NSE in the cerebrospinal fluid or the blood plasma are helpful in detecting neuronal damage in the central nervous system.     

Hypomyelination Phenotype Caused by Impaired Differentiation of Oligodendrocytes in Emx1-cre Mediated Cdk5 Conditional Knockout Mice

Cyclin-dependent kinase 5 (Cdk5) plays a pivotal role in neuronal migration and differentiation, and in axonal elongation. Although many studies have been conducted to analyze neuronal functions of Cdk5, its kinase activity has also been reported during oligodendrocyte differentiation, which suggests Cdk5 may play an important role in oligodendrocytes. Here, we describe a hypomyelination phenotype observed in Emx1-cre mediated Cdk5 conditional knockout (cKO) mice (Emx1-cKO), in which the Cdk5 gene was deleted in neurons, astrocytes and oligodendrocyte -lineage cells. In contrast, the Cdk5 gene in CaMKII cKO mice was deleted only in neurons. Because the development of mature oligodendrocytes from oligodendrocyte precursor cells is a complex process, we performed in situ hybridization using markers for the oligodendrocyte precursor cell and for the differentiated oligodendrocyte. Our results indicate that hypomyelination in Emx1-cKO is due to the impaired differentiation of oligodendrocytes, rather than to the proliferation or migration of their precursors. The present study confirmed the in vivo role of Cdk5 in oligodendrocyte differentiation.     

Oscillatory expression of Ascl1 in oligodendrogenesis

The proneural gene Ascl1 promotes formation of both neurons and oligodendrocytes from neural stem cells (NSCs), but it remains to be analyzed how its different functions are coordinated. It was previously shown that Ascl1 enhances proliferation of NSCs when its expression oscillates but induces differentiation into transit-amplifying precursor cells and neurons when its expression is up-regulated and sustained. By time-lapse imaging and immunohistological analyses, we found that Ascl1 expression oscillated in proliferating oligodendrocyte precursor cells (OPCs) at lower levels than in transit-amplifying precursor cells and was repressed when OPCs differentiated into mature oligodendrocytes. Induction of sustained overexpression of Ascl1 reduced oligodendrocyte differentiation and promoted neuronal differentiation. These results suggest that oscillatory expression of Ascl1 plays an important role in proliferating OPCs during oligodendrocyte formation.     

Characterization of mesenchymal stem cells from rat bone marrow: ultrastructural properties,differentiation potential and immunophenotypic markers

Bone marrow-derived mesenchymal stem cells (BM-MSCs) can differentiate into many lineages. Although the growing interest in BM-MSCs has led to a number of characterization studies, some important biochemical and immunohistochemical properties are still lacking. In this study, morphological and immunophenotypic properties of BM-MSCs were examined in detail. Differentiation potential and growth kinetics of adult rat BM-MSCs were also determined. Immunohistochemistry and RT-PCR results indicated that BM-MSCs expressed myogenic (desmin, myogenin, myosin IIa, and α-SMA), neurogenic (γ-enolase, MAP2a,b, c-fos, nestin, GFAP and beta III tubulin), and osteogenic (osteonectin, osteocalcin, osteopontin, Runx-2, BMP-2, BMP-4 and type I collagen) markers without stimulation towards differentiation. These expression patterns indicated why these cells can easily differentiate into multiple lineages both in vitro and in vivo. Ultrastructural characteristics of rBM-MSCs showed more developed and metabolically active cells.     

Activation of PKC isoforms by ATP/UTP and PMA in murine astrocytes

Abnormal myelin formation appears to be one defect contributing to the neuropathology associated with the fetal alcohol syndrome, the major cause of noncongenital mental retardation. Using the CG-4 cell line we previously showed that 25–75 m m ethanol (EtOH) down-regulates myelin basic protein (MBP) expression in differentiating oligodendrocytes (OLGs) without affecting the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) expression or morphological development (Bichenkov and Ellingson 2001). Here we observed that a relatively low concentration of 12-phorbol-13-myristate acetate (PMA) mimicked the EtOH-caused inhibition of MBP expression without affecting CNP expression or morphology. The inhibition of MBP expression by 100 m m EtOH or 1 n m PMA was completely counteracted by three inhibitors of protein kinase C (PKC); bisinodoylmaleimide I, chelerythrine chloride, and calphostin C, indicating that EtOH down-regulated MBP expression by activating PKC. We investigated whether the EtOH-caused activation resulted in part from up-regulation of the expression of PKC isozymes. Of 11 PKC isozymes examined, CG-4 OLGs expressed nine; PKC α, β1, β2, δ, ε, λ, μ, nu and zeta; while PKC isozymes γ and theta were not detected. Only five PKC isozymes, α, β1, β2, μ, and nu, displayed developmental changes in expression. However, EtOH did not up-regulate the early expression of any PKC isozyme during the first two days of differentiation, the developmental stage when it down-regulates the MBP expression in CG-4 cells. The results indicate that EtOH delays MBP expression by activating at least one phorbol ester-sensitive PKC isozyme in differentiating oligodendrocytes without up-regulating its expression.
Acknowledgements:   Support: NIAAA Grant AA072185.       

Up-regulation of E-cadherin and β-catenin in human hepatocellular carcinoma cell lines by sodium butyrate and interferon-α

Summary Human E-cadherin is a homophilic cell adhesion molecule and its expression is well preserved in normal human hepatocytes; a decrease in its expression has been observed in poorly differentiated hepatocellular carcinoma cells. We examined the alteration of E-cadherin and catenin expressions caused by differentiation inducers in human hepatocellular carcinoma cells. Hepatocellular carcinoma cell lines, HCC-T and HCC-M, were cultured with all-trans retinoic acid (ATRA), dexamethasone (DEX), sodium butyrate, and interferon-α. E-cadherin expression was only up-regulated by butyrate and interferon-α (IFN-α) in both cell lines, studied by means of fluorescence immunostaining and flow cytometry. The localization of E-cadherin staining was shown at their cell membrane. According to the increase in E-cadherin expression, β-catenin expression appeared at the cell membrane of both cell lines when treated with butyrate and IFN-α. Such an appearance was not observed when cells were treated with ATRA and DEX. Western blotting showed that α-and γ-catenin expression was not changed, while only the expression of β-catenin increased. β-Catenin oncogenic activation as a result of amino acid substitutions or interstitial deletions within or including parts of exon 3, which has been demonstrated recently, was not detected in these cell lines by direct deoxyribonucleic acid sequencing. These results suggest that the expression and interaction between E-cadherin and wild-type β-catenin are potentially modulated by butyrate and IFN-α, and that these two agents are potent inhibitors of hepatocellular carcinoma cell invasion and metastasis.     

Oligodendrocytes are a Novel Source of Amyloid Peptide Generation

Alzheimer’s disease is characterised by regional neuronal degeneration, synaptic loss, and the progressive deposition of the 4 kDa β-amyloid peptide (Aβ) in senile plaques and accumulation of tau protein as neurofibrillary tangles. Aβ derives from the larger precursor molecule, amyloid precursor protein (APP) by proteolytic processing via β- and γ-secretases. While APP expression is well documented in neurons and astrocytes, the case for oligodendrocytes is less clear. The latter cell type is reported to express different isoforms of APP, and we have confirmed this observation by immunocytochemistry in cultures of differentiated rat cortical oligodendrocytes. Moreover, by means of a sensitive electrochemiluminescent immunoassay employing Aβ C-terminal specific antibodies, mature oligodendrocytes are shown to secrete the 40 and 42 amino acid Aβ species (Aβ40 and Aβ42). Secretion of Aβ peptides was reduced by incubating oligodendrocytes with α- and β-secretase inhibitors, or a γ-secretase inhibitor. Disturbances of APP processing and/or synthesis in oligodendrocytes may account for some myelin disorders observed in Alzheimer’s disease and other senile dementias.     

Evidence that GABAA Receptor Subunit mRNA Expression During Development Is Regulated by GABAA Receptor Stimulation

Abstract: The expression of six mRNA species (α2, α3, α5, β2, β3, and γ2) encoding for GABA_A receptor subunits was followed in cultured early postnatal cortical neurons by in situ hybridization histochemistry. In untreated control cultures it was found that these subunit mRNA expression profiles closely follow those seen during development in vivo. α3, α5, and β3 subunit expression declined, α2 expression increased, whereas β2 and γ2 subunit mRNA expression remained relatively constant. To test the hypothesis that GABA_A receptor stimulation regulates these expression profiles, we tested the effect of a GABA_A receptor positive modulator, allopregnanolone, and a GABA_A receptor noncompetitive antagonist, tert -butylbicyclophosphorothionate (TBPS). It was found that allopregnanolone augmented the rate at which the α3, α5, or β3 subunit mRNA expression declined and prevented the increase in α2 subunit mRNA expression. As well, allopregnanolone down-regulated β2 subunit mRNA expression. TBPS, on the other hand, up-regulated α3, α5, β2, and β3 subunit mRNA expression. It also down-regulated the expression of α2 subunit mRNA. Both allopregnanolone and TBPS had no effect on γ2 subunit mRNA expression. These results imply that the developmental switchover of GABA receptor subunit mRNA expression is regulated by GABA_A receptor activity.