PRMT1 negatively regulates activation-induced cell death in macrophages by arginine methylation of GAPDH.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is implicated in cell death in addition to a role as a glycolytic enzyme. In particular, when cells are exposed to cellular stressors involving nitric oxide (NO) production, GAPDH can undergo NO-induced S-nitrosylation and S-nitrosylated GAPDH has been shown to elicit apoptosis. However, the mechanism underlying the regulation of the pro-apoptotic function of GAPDH remains unclear. Here, we found that protein arginine methyltransferase 1 (PRMT1) mediated arginine methylation of GAPDH in primary bone marrow-derived macrophages in a NO-dependent manner. Moreover, PRMT1 inhibited S-nitrosylation of GAPDH as well as its binding to SIAH1, thereby reducing the nuclear translocation of GAPDH in lipopolysaccharide (LPS)/interferon (IFN)-γ-activated macrophages. Furthermore, depletion of PRMT1 expression by RNA interference potentiated LPS/IFN-γ-induced apoptosis in macrophages. Taken together, our results suggest that PRMT1 has a previously unrecognized function to inhibit activation-induced cell death of macrophages through arginine methylation of GAPDH.

Selective glucocorticoid receptor modulator compound A, in contrast to prednisolone, does not induce leptin or the leptin receptor in human osteoarthritis synovial fibroblasts.

OBJECTIVE: Glucocorticoids are powerful anti-inflammatory compounds that also induce the expression of leptin and leptin receptor (Ob-R) in synovial fibroblasts through TGF-ßsignalling and Smad1/5 phosphorylation. Compound A (CpdA), a selective glucocorticoid receptor agonist, reduces inflammation in murine arthritis models and does not induce diabetes or osteoporosis, thus offering an improved risk:benefit ratio in comparison with glucocorticoids. Due to the detrimental role of leptin in OA pathogenesis, we sought to determine whether CpdA also induced leptin and Ob-R protein expression as observed with prednisolone. METHODS: Human synovial fibroblasts and chondrocytes were isolated from the synovium and cartilage of OA patients after joint surgery. The cells were treated with prednisolone, TGF-ß1, TNF-α and/or CpdA. Levels of leptin, IL-6, IL-8, MMP-1 and MMP-3 were measured by ELISA and expression levels of Ob-R phospho-Smad1/5, phospho-Smad2, α-tubulin and glyceraldehyde 3-phosphate dehydrogenase were analysed by western blotting. RESULTS: CpdA, unlike prednisolone, did not induce leptin secretion or Ob-R protein expression in OA synovial fibroblasts. Moreover, CpdA decreased endogenous Ob-R expression and down-regulated prednisolone-induced leptin secretion and Ob-R expression. Mechanistically, CpdA, unlike prednisolone, did not induce Smad1/5 phosphorylation. CpdA, similarly to prednisolone, down-regulated endogenous and TNF-α-induced IL-6, IL-8, MMP-1 and MMP-3 protein secretion. The dissociative effect of CpdA was confirmed using chondrocytes with no induction of leptin secretion, but with a significant decrease in IL-6, IL-8, MMP-1 and MMP-3 protein secretion. CONCLUSION: CpdA, unlike prednisolone, did not induce leptin or Ob-R in human OA synovial fibroblasts, thereby demonstrating an improved risk:benefit ratio.

An aggregate-prone mutant of human glyceraldehyde-3-phosphate dehydrogenase augments oxidative stress-induced cell death in SH-SY5Y cells.

Glycerladehyde-3-phosphate dehydrogenase (GAPDH), a classic glycolytic enzyme, also has a role in mediating cell death under oxidative stress. Our previous reports suggest that oxidative stress-induced GAPDH aggregate formation is, at least in part, a mechanism to account for the death signaling. Here we show that substitution of cysteine for serine-284 of human GAPDH (S284C-GAPDH) leads to aggregate-prone GAPDH, and that its expression in SH-SY5Y human neuroblastoma results in greater dopamine-induced cell death than expression of wild type-GAPDH. Treatment of purified recombinant S284C-GAPDH in vitro with the nitric oxide donor NOR3 led to greater aggregation than wild type-GAPDH. Several lines of structural analysis revealed that S284C-GAPDH was amyloidogenic. Overexpression of doxycycline-inducible S284C-GAPDH in SH-SY5Y cells accelerated dopamine treatment-induced death and increased formation of GAPDH aggregates, compared to cells expressing wild type-GAPDH. These results suggest that aggregate-prone mutations of GAPDH such as S284C-GAPDH may confer risk of oxidative stress-induced cell death.

PI3K/Akt mediates expression of TNF-alpha mRNA and activation of NF-kappaB in calyculin A-treated primary osteoblasts.

OBJECTIVE: The effect of calyculin A (CA), a serine/threonine protein phosphatase inhibitor, on tumor necrosis factor-alpha (TNF-alpha) in primary osteoblasts was investigated to determine whether protein phosphatases could affect primary osteoblasts and if so which signaling pathways would be involved. MATERIALS AND METHODS: Primary osteoblasts were prepared from newborn rat calvaria. Cells were treated with 1 nM CA for different time periods. The expressions of TNF-alpha and GAPDH mRNA were determined by RT-PCR. Cell extracts were subjected to SDS-PAGE and the activation of Akt and NF-kappaB were analyzed by western blotting. RESULTS: Calyculin A-treatment markedly increased the expression of TNF-alpha mRNA and enhanced the phosphorylation level of Akt (Ser473) in these cells. Pretreatment with the PI3K inhibitor LY294002 suppressed the increase in TNF-alpha mRNA expression and the phosphorylation of Akt in response to CA. Western blot analysis showed that CA stimulated the phosphorylation and nuclear translocation of NF-kappaB in primary osteoblasts, and these responses were blocked by pretreatment with LY294002. CONCLUSION: Calyculin A elicits activation of PI3K/Akt pathway which leads to expression of TNF-alpha mRNA and activation of NF-kappaB. This NF-kappaB activation involves both phosphorylation and nuclear translocation of NF-kappaB.

Retinoic acid attenuates the mild hyperoxic lung injury in newborn mice.

Neonatal exposure to hyperoxia alters lung development in mice. We tested if retinoic acid (RA) treatment is capable to affect lung development after hyperoxic injury and to maintain structural integrity of lung. The gene of vascular endothelial growth factor A (VEGF-A) is one of the RA-responsive genes. Newborn BALB/c mice were exposed to room air, 40% or 80% hyperoxia for 7 days. One half of animals in each group received 500 mg/kg retinoic acid from day 3 to day 7 of the experiment. At the end of experiment we assessed body weight (BW), lung wet weight (LW), the wet-to-dry lung weight ratio (W/D) and the expression of mRNA for VEGF-A and G3PDH genes. On day 7 the hyperoxia-exposed sham-treated mice (group 80) weighed 20% less than the room air-exposed group, whereas the 80% hyperoxic group treated with RA weighed only 13% less than the normoxic group. W/D values in 80 and 80A groups did not differ, although they both differed from the control group and from 40 groups. There was a significant difference between 40 and 40A groups, but the control group was different from 40 group but not from 40A groups. The 80 and 80A groups had mRNA VEGF-A expression lowered to 64% and 41% of the control group. RA treatment of normoxic and mild hyperoxic groups increased mRNA VEGF-A expression by about 50%. We conclude that the retinoic acid treatment of newborn BALB/c mice exposed for 7 days to 80% hyperoxia reduced the growth retardation in the 80 % hyperoxic group, reduced the W/D ratio in the 40% but not in the 80 % hyperoxic group. Higher VEGF-A mRNA expression in the 80% hyperoxic group treated with RA was not significant compared to the 80% hyperoxic group.

Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth.

The aim of the study was to evaluate the effects of the capping materials mineral trioxide aggregate (MTA), calcium hydroxide (CH) and BiodentineTM (BD) on stem cells from human exfoliated deciduous teeth (SHED) in vitro. SHED were cultured for 1 - 7 days in medium conditioned by incubation with MTA, BD or CH (1 mg/mL), and tested for viability (MTT assay) and proliferation (SRB assay). Also, the migration of serum-starved SHED towards conditioned media was assayed in companion plates, with 8 µm-pore-sized membranes, for 24 h. Gene expression of dentin matrix protein-1 (DMP-1) was evaluated by reverse-transcription polymerase chain reaction. Regular culture medium with 10% FBS (without conditioning) and culture medium supplemented with 20% FBS were used as controls. MTA, CH and BD conditioned media maintained cell viability and allowed continuous SHED proliferation, with CH conditioned medium causing the highest positive effect on proliferation at the end of the treatment period (compared with BD and MTA) (p<0.05). In contrast, we observed increased SHED migration towards BD and MTA conditioned media (compared with CH) (p<0.05). A greater amount of DMP-1 gene was expressed in MTA group compared with the other groups from day 7 up to day 21. Our results show that the three capping materials are biocompatible, maintain viability and stimulate proliferation, migration and differentiation in a key dental stem cell population.

Thalidomide suppresses melanoma growth by activating natural killer cells in mice.

Although thalidomide (Thd) is being extensively investigated for its effects on cytokine production and T cell costimulation, it is poorly understood whether it is capable of modulating the activities of natural killer (NK) cells. In this study, Thd effects on NK cell activity were examined with a murine model of melanoma, which is mostly rejected by NK cell-dependent mechanism. Administration of Thd significantly (p<0.01 on Day 21) suppressed the growth of subcutaneous B16F1 melanoma. In Thd-treated mice, marked splenomegaly and augmented splenocyte count were observed. Additionally, the percentage of splenic NK1.1+ cells was elevated to approximately 2.5-fold within 10 days after Thd treatment. The expression of interferon inducible protein (IP)-10, interferon (IFN)-gamma, interleukin (IL)-12 and IL-18 was remarkably upregulated. Production of the cytotoxic molecule perforin was also augmented. These data suggest that Thd strongly activates NK cell activity in mice, possibly resulting in enhanced tumor surveillance defense.

A nuclear protein complex containing high mobility group proteins B1 and B2, heat shock cognate protein 70, ERp60, and glyceraldehyde-3-phosphate dehydrogenase is involved in the cytotoxic response to DNA modified by incorporation of anticancer nucleoside analogues.

Thiopurine treatment of human leukemia cells deficient in components of the mismatch repair system (Nalm6) initiated apoptosis after incorporation into DNA, as revealed by caspase activation and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. To elucidate the cellular sensor(s) responsible for recognition of DNA damage in cells with an inactive mismatch repair system, we isolated a multiprotein nuclear complex that preferentially binds DNA with thioguanine incorporated. The components of this nuclear multiprotein complex, as identified by protein mass spectroscopy, included high mobility group proteins 1 and 2 (HMGB1, HMGB2), heat shock protein HSC70, protein disulfide isomerase ERp60, and glyceraldehyde 3-phosphate dehydrogenase. The same complex was also shown to bind synthetic oligodeoxyribonucleotide duplexes containing the nonnatural nucleosides 1-beta-D-arabinofuranosylcytosine or 5-fluoro-2'-deoxyuridine. Fibroblast cell line derived from Hmgb1(-/-) murine embryos had decreased sensitivity to thiopurines, with an IC(50) 10-fold greater than Hmgb1-proficient cells (P < 0.0001) and exhibited comparable sensitivity to vincristine, a cytotoxic drug that is not incorporated into DNA. These findings indicate that the HMGB1-HMGB2-HSC70-ERp60-glyceraldehyde 3-phosphate dehydrogenase complex detects changes in DNA structure caused by incorporation of nonnatural nucleosides and is a determinant of cell sensitivity to such DNA modifying chemotherapy.

[Molecular mechanisms of the neuroprotective effect of (-)-deprenyl]. / A (-)-deprenyl neuroprotektív hatásának molekuláris mechanizmusai.

(-)-Deprenyl, the irreversible inhibitor of monoamine oxidase B, has been used for decades in the therapy of Parkinson's disease. It improves parkinsonian symptoms due to its dopamine potentiating and antioxidant properties and presumedly delays disease progression. Its complex pharmacological action cannot be explained solely by its monoamine oxidase B inhibitory property. Recently, (-)-deprenyl has been demonstrated to exert antiapoptotic, neuroprotective effects on a number of in vitro and in vivo models in a dose significantly lower than required for monoamine oxidase B inhibition. (-)-Deprenyl and related propargylamines prevent apoptotic cell death by preserving the integrity of the mitochondrion that may be based on the activation of a complex transcriptional program. The changes in gene expression initiated by propargylamines incited to search for further possible target molecules that would explain more accurately the antiapoptotic effect of these compounds. The latest molecular targets include such classical metabolic enzymes, the homologues of which may participate in the regulation of gene expression as a part of transcriptional factor complexes. Some of the propargylamine targets--glyceraldehyde-3-phosphate dehydrogenase, poly(ADP-ribose) polymerase, nuclear amine oxidases--have already been demonstrated to be capable of transforming the metabolic changes in the cell to transcriptional responses. Data are accumulating about the relationship of these enzymes and propargyl compounds, but the real significance of this issue will only be established by future research.

C-terminal truncation of spinach chloroplast NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase prevents inactivation and reaggregation.

Chloroplast NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) consists of two types of subunits: GapA and GapB, which are rather similar, except that GapB carries an unique C-terminal sequence extension. Here, we report evidence that this sequence extension might be responsible for aggregation and dark inactivation of the enzyme in vivo. Recently, it had been demonstrated that upon limited proteolysis of the purified 600 kDa enzyme, using the Staphylococcus aureus V8 endoproteinase (Zapponi et al. (1993) Biol. Chem. Hoppe-Seyler 374, 395-402), the C-terminus of GapB can be removed, giving rise to the 150 kDa form. Based on these findings, we analyzed the changed catalytic properties of the enzyme after proteolysis and its ability to reaggregate. The time-course of proteolysis is paralleled by a strong increase in enzyme activity and the appearance of the tetrameric enzyme form, the increase of apparent activity preceding disaggregation. The proteolyzed enzyme is characterized by its increased affinity towards the substrate 1,3-bisphosphoglycerate and thus resembles the fully activated intact enzyme. In contrast to the effector-mediated activation of the intact enzyme, both proteolytic activation and the resulting disaggregation of the high-molecular-weight form cannot be reversed, even by incubation with NAD.

An asynchronous unfolding among molecular different regions of lobster D-glyceraldehyde-3-phosphate dehydrogenase and maltotetraose-forming amylase from an Alcaligenes sp. during guanidine denaturation.

Changes in ultraviolet absorbance and intrinsic protein fluorescence of 1,4-alpha-D-glucan maltotetrahydrolase (EC 3.2.1.60) from an Alcaligenes sp. (Gram-negative bacteria 537.1) and D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) have been compared with their inactivation during denaturation in guanidinium-Cl solutions. The two enzymes were completely inactivated at GuHCl concentrations less than 0.6 M and this was accompanied by marked absorbance and intrinsic fluorescence changes suggesting exposure of aromatic residues. The changes of the intrinsic fluorescence of the amylase have a relatively constant plateau in emission intensities and maxima at GuHCl concentrations from 0.8-2.0 M, similar to that of muscle GAPDH. The relative activity of the enzyme increased markedly in dilute GuHCl solutions accompanied by very little change of its intrinsic fluorescence at 8 degrees C. The kinetic decrease in emission intensities, excited respectively by 230 nm and 292 nm, was different for the two enzymes. The inactivation was a biphasic process with a fast phase faster than the unfolding rate as measured by fluorescence changes in 0.5 M GuHCl solution. Similar to the inactivation process, changes in intensity of 410 nm NAD fluorescent derivative of GAPDH which is in situ at the active site is also a biphasic process under the same condition. It appears that there may be an unfolding intermediate state of the enzymes and an asynchronous unfolding process among the different regions in the molecules during GuHCl denaturation, this may be due to differences in their flexibility.

Rat brain glycolysis regulation by estradiol-17 beta.

The effect of estradiol-17 beta on the activities of glycolytic enzymes from female rat brain was studied. The following enzymes were examined: hexokinase (HK, EC 2.7.1.1), phosphofructokinase (PFK, EC 2.7.1.11), aldolase (EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), phosphoglycerate mutase (EC 2.7.5.3), enolase (EC 4.2.1.11) and pyruvate kinase (PK, EC 2.7.1.40). The activities of HK (soluble and membrane-bound), PFK and PK were increased after 4 h of hormone treatment, while the others remained constant. The changes in activity were not seen in the presence of actinomycin D. The significant rise of the activities of the key glycolytic enzymes was also observed in the cell culture of mouse neuroblastoma C1300 treated with hormone. Only three of the studied isozymes, namely, HKII, B4 and K4 were found to be estradiol-sensitive for HK, PFK and PK, respectively. The results obtained suggest that rat brain glycolysis regulation by estradiol is carried out in neurons due to definite isozymes induction.

Methylglyoxal can modify GAPDH activity and structure.

The activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can play an important role in regulating multiple upstream pathways relating to the development of diabetic complications. GAPDH can be modified by a number of metabolic factors, including oxidative and glycation products. To study the effect of glycation on GAPDH we have measured GAPDH structure and activity after exposure of the enzyme to the potent alpha dicarbonyl sugar methylglyoxal (MG). Rabbit GAPDH was incubated with 10-1000 microM MG for 96 hours, and enzyme activity was measured at intervals by a spectrophotometric assay. Isoelectric focusing of purified and cellular GAPDH was performed with a PROTEAN IEF system and the bands visualized by Western blotting. The mass of glycated and native GAPDH was determined by MALDI with a Applied Biosystems Voyager System 6235. GAPDH activity (at 96 h) was decreased by 20% with 1.0 micromolar MG and showed progressively greater suppression of activity with increasing concentrations up to 1 mM, where activity was decreased by 97%. Reduction in GAPDH activity was rapidly decreasing by 69.2% by two hours with 1 mM MG. IEF showed an isoelectric point (IEP) of 8.5 for native GAPDH, while measurable changes were seen with modification by MG levels of 1 mM (IEP 7.5) and 50 microM (IEP 8.0). With MALDI, GAPDH mass increased from 36.012 kDa to 37.071 after exposure to 50 microM MG and to 40.625 following 1 mM MG. This indicates addition of 12.75 and 55.6 MG residues, respectively, to GAPDH. GAPDH can be modified by methylglyoxal intracellular concentrations close to those previously observed in vivo, with measurable changes in isoelectric point and mass. These modifications can lead to decreased enzyme activity, suggesting that conditions associated with elevated intracellular MG could modify GAPDH activity in vivo.

RANKL expression is related to the differentiation state of human osteoblasts.

Human osteoblast phenotypes that support osteoclast differentiation and bone formation are not well characterized. Osteoblast differentiation markers were examined in relation to RANKL expression. RANKL expression was induced preferentially in immature cells. These results support an important link between diverse osteoblast functions. Cells of the osteoblast lineage support two apparently distinct functions: bone formation and promotion of osteoclast formation. The aim of this study was to examine the relationship between these phenotypes in human osteoblasts (NHBC), in terms of the pre-osteoblast marker, STRO-1, and the mature osteoblast marker, alkaline phosphatase (AP), and the expression of genes involved in osteoclast formation, RANKL and OPG. The osteotropic stimuli, 1alpha,25(OH)2vitamin D3 (vitD3) and dexamethasone, were found to have profound proliferative and phenotypic effects on NHBCs. VitD3 inhibited NHBC proliferation and increased the percentage of cells expressing STRO-1 over an extended culture period, implying that vitD3 promotes and maintains an immature osteogenic phenotype. Concomitantly, RANKL mRNA expression was upregulated and maintained in NHBC in response to vitD3. Dexamethasone progressively promoted the proliferation of AP-expressing cells, resulting in the overall maturation of the cultures. Dexamethasone had little effect on RANKL mRNA expression and downregulated OPG mRNA expression in a donor-dependent manner. Regression analysis showed that RANKL mRNA expression was associated negatively with the percentage of cells expressing AP (p < 0.01) in vitD3- and dexamethasone-treated NHBCs. In contrast, RANKL mRNA expression was associated positively with the percentage of STRO-1+ cells (p < 0.01). In NHBCs sorted by FACS based on STRO-1 expression (STRO-1bright and STRO-1dim populations), it was found that vitD3 upregulated the expression of RANKL mRNA preferentially in STRO-1bright cells. The results suggest that immature osteoblasts respond to osteotropic factors in a potentially pro-osteoclastogenic manner. Additionally, the dual roles of osteoblasts, in supporting osteoclastogenesis or forming bone, may be performed by the same lineage of cells at different stages of their maturation.

Reversible nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase upon serum depletion.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12) functions as a glycolytic enzyme within the cytoplasm, but beside its metabolic function it is involved in early steps of apoptosis, which trigger the translocation of GAPDH into the nucleus. As apoptosis can be induced by serum withdrawal, which otherwise causes cell cycle arrest, the linkage between serum deprivation, cell cycle and nuclear transport of GAPDH has been investigated. The intracellular distribution of GAPDH was monitored by confocal laser scanning microscopy of either immuno-stained NIH 3T3 fibroblasts or of cells overexpressing GFP-tagged GAPDH. Serum withdrawal led to an accumulation of GAPDH in the nucleus. In contrast to investigations published so far, this nuclear translocation was a reversible process: cytoplasmic location of endogenous GAPDH or of GFP-GAPDH could be recovered upon serum addition to arrested cells and was not inhibited by cycloheximide treatment. In addition, the nuclear import upon serum depletion had no influence neither on the catalytic activity nor on the expression level of GAPDH. The nuclear export of GFP-GAPDH in serum-deprived cells could be stimulated by serum or directly by the growth factors EGF or PDGE The transport process is not regulated via an initiation of cell cycle arrest, as olomoucine, which causes G1-arrest neither stimulated nuclear accumulation nor prevented nuclear export after serum addition to serum-depleted cultures. Moreover, SV40-transformed 3T3 cells transported GAPDH into the nucleus upon serum deprivation, though the expression of the viral large T-antigen enabled growth factor-independent cell proliferation in this cell line. The recruitment of GAPDH to the cytoplasm upon serum stimulation of arrested cells was not impaired by the inhibition of the MAPK signalling pathway with PD 098059. However, further analysis of the growth factor signalling pathway with specific inhibitors revealed that nuclear export was prevented by LY 294002, an inhibitor of the PI-3 kinase. PI3K links the growth factor signalling pathway with cell death via the repression of an apoptotic inducer. Thus, the nuclear accumulation of GAPDH upon growth factor depletion is a reversible process not related directly to cell cycle and likely triggered by survival signals.

Quantitative histochemistry of the primate skin. III. Glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase activities were assayed with a fluorometric micromethod in various parts of the skin and its appendages. Enzyme activity was abundant, particularly in the epidermis and mucous membrane (4.2 to 11.3 moles/hr/kg dry wt.). The keratin layer of sole epidermis also contained a considerable amount of enzyme activity (2.6 moles/hr/kg dry wt.). A relatively low activity was found in the apocrine and sebaceous glands (1.8 to 3.6 moles/hr/kg dry wt.). The enzyme was influenced by various activators and inhibitors. The activators were 1-cysteine (200% increase), mercaptoethanol (+300%), EDTA (+300%), and the inhibitors, pCMB, Cu++ and Co++ (nearly 100% inhibition). Either arsenate or orthophosphate was an absolute requirement for the enzyme reaction. It is speculated that this dehydrogenase in skin and appendages may be a regulator of the Pasteur effect, hence of glycolysis, as it may be in muscle or in ascites tumor cells.

Regulation of glyceraldehyde-3-phosphate dehydrogenase in differentiating HD3 cells.

Red blood cells usually replenish their ATP pools by glycolysis, fueled by glucose imported via the cell membrane. Mature red cells of some species (e.g. pig, chicken) have, however, been reported to show very low glucose transport. The subject of this study was the possible dependency of the level of a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAD) on glucose transporter activity during the maturation of chicken red cells. The chicken pronormoblast cell line, HD3, was used as a model system. These cells contain higher levels of GAD and glucose transporter activities than normal chicken bone marrow cells, but reduce their levels during maturation. In an attempt to assess whether the decrease in GAD activity is regulated by the glucose transport, the chicken GLUT3 expressed under the control of viral promoter was introduced into HD3 cells by retroviral infection (pDOL-cGT3). Upon cell differentiation and maturation, both cells with and without the exogenous transporter decreased GAD activity. Butyric acid did not affect the regulation of GAD activity upon differentiation. These results show that the development of chicken red cells is manifested by reduction of their GAD activity and that this is not affected by their sugar transporter activity. The very low GAD activity in embryonic chicken red cells is thus due to a loss of this activity at an early stage in their development. Because of the very low glucose transport and GAD activities in mature chicken red cells, rates of glycolysis are likely to be low and suggesting an alternative pathway for ATP production in these cells.

A selective effect of Mpl ligand on mRNA stabilization during megakaryocyte differentiation.

Megakaryocytes, the platelet precursors, are induced to differentiate in response to Mpl ligand. Here we report that stability of the megakaryocyte-specific platelet factor 4 (PF4) mRNA is substantially augmented in the presence of Mpl ligand. This stabilization requires protein synthesis, but the 3'-untranslated region of PF4 mRNA is not sufficient for granting the effect. This cytokine also significantly or mildly stabilizes Mpl receptor or GAPDH mRNAs, respectively, in contrast to a previously reported lack of effect on P2Y(1) receptor mRNA. Our study is the first to suggest that Mpl ligand-induced lineage specification is also determined by message stabilization.

Alpha-lipoic acid protects the retina against ischemia-reperfusion.

The aim of this study was to examine whether the antioxidant alpha-lipoic acid protects retinal neurons from ischemia-reperfusion injury. Rats were injected intraperitoneally with either vehicle or alpha-lipoic acid (100 mg/kg) once daily for 11 days. On the third day, ischemia was delivered to the rat retina by raising the intraocular pressure above systolic blood pressure for 45 min. The electroretinogram was measured prior to ischemia and 5 days after reperfusion. Rats were killed 5 or 8 days after reperfusion and the retinas were processed for immunohistochemistry and for determination of mRNA levels by RT-PCR. Ischemia-reperfusion caused a significant reduction of the a- and b-wave amplitudes of the electroretinogram, a decrease in nitric oxide synthase and Thy-1 immunoreactivities, a decrease of retinal ganglion cell-specific mRNAs and an increase in bFGF and CNTF mRNA levels. All of these changes were clearly counteracted by alpha-lipoic acid. Moreover, in mixed rat retinal cultures, alpha-lipoic acid partially counteracted the loss of GABA-immunoreactive neurons induced by anoxia. The results of the study demonstrate that alpha-lipoic acid provides protection to the retina as a whole, and to ganglion cells in particular, from ischemia-reperfusion injuries. alpha-Lipoic acid also displayed negligible affinity for voltage-dependent sodium and calcium channels.

Norepinephrine, tri-iodothyronine and insulin upregulate glyceraldehyde-3-phosphate dehydrogenase mRNA during Brown adipocyte differentiation.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was studied in differentiating brown adipocytes. Northern blot analysis showed that GAPDH mRNA levels increased during differentiation of precursor cells into mature adipocytes, mainly in the initial stages of the differentiation process. Insulin, tri-iodothyronine (T(3)) and norepinephrine, the main regulators of brown adipose tissue function, upregulated GAPDH mRNA levels, whereas retinoic acid inhibited them. The effect of insulin was present on all culture days examined, was time- and dose-dependent, and was exerted through its own receptors, as demonstrated by comparing insulin and insulin-like growth factor (IGF)-I and -II potencies in this system. Using the transcriptional inhibitor, actinomycin D, we demonstrated that T(3), and to a lesser extent insulin, stabilized GAPDH mRNA. Experiments with cycloheximide indicated that both hormones require de novo protein synthesis to achieve their effects. Using cAMP analogs, we showed that the effect of norepinephrine is probably exerted through this second messenger. Co-operation was elucidated between norepinephrine- and insulin-mediated induction of GAPDH mRNA levels. In summary, we have demonstrated that GAPDH mRNA is subjected to multifactorial regulation in differentiating brown adipocytes that includes differentiation of precursor cells and the lipogenic/lipolytic regulators of the tissue.