Cloning, expression and characterization of a UDP-galactose 4-epimerase from Escherichia coli

The gene galE encoding UDP-galactose 4-epimerase was cloned into E. coli BL21(DE3) from the chromosomal DNA of E. coli strain K-12. High expression of the soluble recombinant epimerase was achieved in the cell lysate. In order to evaluate the use of this epimerase in enzymatic synthesis of important -Gal epitopes (oligosaccharides with a terminal Gal1,3Gal sequence), a new radioactivity assay (1,3-galactosyltransferase coupled assay) was established to characterize its activity in producing UDP-galactose from UDP-glucose. Approximately 2700 units (100 mg) enzyme with a specific activity of 27 U mg^–1 protein could be obtained from one liter of bacterial culture. The epimerase was active in a wide pH range with an optimum at pH 7.0. This expression system established a viable route to the enzymatic production of -Gal oligosaccharides to support xenotransplantation research.     

内皮素-1促进反应性星形胶质细胞增殖

目的利用成年SD大鼠脊髓损伤原代培养的反应性星形胶质细胞模型,探讨内皮素-1(ET1)与反应性星形胶质细胞增殖之间的关系。方法建立成年SD大鼠脊髓损伤原代培养的反应性星形胶质细胞模型,用100 n M ET1和5μM BQ788(内皮素受体B的拮抗剂)处理反应性星形胶质细胞48 h,通过免疫荧光的方法对各实验组中星形胶质细胞的标记分子Vimentin及Brdu进行检测,以确定ET1对反应性星形胶质细胞增殖的影响。结果 ET1组中星形胶质细胞的数量明显增加,Brdu阳性细胞占星形胶质细胞的平均百分比(19.41%)高于正常对照组(3.28%,P0.01);而ET1+BQ788组中Brdu阳性细胞数占星形胶质细胞的平均百分比为10.38%,明显低于ET1组(19.41%,P0.01)。结论在成年SD大鼠脊髓损伤原代培养的反应性星形胶质细胞模型中,ET1可刺激反应性星形胶质细胞的增殖,ET1受体endothelin B的拮抗剂BQ788可有效抑制ET1对反应性星形胶质细胞的促增殖效应。     

The blockade of endothelin A receptor protects astrocytes against hypoxic injury: common effects of BQ-123 anderythropoietin on the rejuvenation of the astrocyte population

In the present study the role of endothelin (ET) and its receptors (ETA-R and ETB-R) in cellular mechanisms underlying the resistance of astroglial cells to low oxygen level and development of hypoxia has been investigated. To define the influences of ET and its receptors on survival and on antigenic as well as morphologic differentiation of rat astroglial cells in normoxic (NC) and hypoxic culture (HC) the selective antagonists of ETA-R (BQ-123) and ETB-R (BQ-788) were used. Treatment of HC with BQ-123 caused an increase in cell number and inhibited the hypoxia-induced apoptosis by 37%. BQ-123 decreased the hypoxia-induced cytotoxicity in HC. These effects of BQ-123 were abolished in cultures simultaneously treated with BQ-123 and BQ-788. Administration of BQ-788 alone decreased the number of living cells in NC, but not in HC. The activity of caspase-3/-7 was not changed by exposure of NC and HC to BQ-788. The protection provided by BQ-123 to astroglial cells against cytotoxicity in NC and HC was similar to that of erythropoietin (EPO), a cytokine with established neuroprotective effects. The functional improvement of astroglial cells and slowing down of their differentiation under exposure to BQ-123, or EPO, or BQ-123 + EPO has been evidenced by an increased number of nestin+/glial fibrillary acidic protein-positive (GFAP+) astrocytes accompanied by decrease of nestin-/GFAP+ cells. The simultaneous treatment with BQ-123 and EPO additionally decreased the activities of caspase-3/-7 (64%) and release of LDH into the medium (94%). The benefits in the functional states of astrocytes obtained by combined treatment of HC with BQ-123 and EPO suggest a new therapeutic strategy in treatment of hypoxic brain injury.     

Structural determinants for selective recognition of peptide ligands for endothelin receptor subtypes ETA and ETB

The molecular basis for recognition of peptide ligands endothelin‐1, ‐2 and ‐3 in endothelin receptors is poorly understood. Especially the origin of ligand selectivity for ET_A or ET_B is not clearly resolved. We derived sequence‐structure‐function relationships of peptides and receptors from mutational data and homology modeling. Our major findings are the dissection of peptide ligands into four epitopes and the delineation of four complementary structural portions on receptor side explaining ligand recognition in both endothelin receptor subtypes. In addition, structural determinants for ligand selectivity could be described. As a result, we could improve the selectivity of BQ3020 about 10‐fold by a single amino acid substitution, validating our hypothesis for ligand selectivity caused by different entrances to the receptors' transmembrane binding sites. A narrow tunnel shape in ET_A is restrictive for a selected group of peptide ligands' N‐termini, whereas a broad funnel‐shaped entrance in ET_B accepts a variety of different shapes and properties of ligands. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Bcl-2 blocks apoptotic signal of transforming growth factor-β in human hepatoma cells

Transforming growth factor- (TGF-) has been shown to induce apoptosis on normal hepatocytes and hepatoma cells both in vitro and in vivo. However, how the TGF- induces apoptosis is still not clear. We examined the expression of anti-apoptosis proteins and sensitivity to TGF- in three well differentiated human hepatoma cell lines. Two TGF- sensitive cell lines Hep3B and HuH7 totally lacked Bcl-2. In contrast, the TGF- resistant HepG2 cells expressed a substantial amount of Bcl-2. All three cell lines expressed equal amounts of Bcl-X_L, Bcl-X_S and Bax. Overexpression of Bcl-2 in Hep3B and HuH7 cells protected them from TGF--induced apoptosis. TGF- treatment increased intracellular peroxide production and suppressed the expression of glutathione-S-transferase in the Hep3B cells, and these effects were partially suppressed by the overexpression of Bcl-2. These results suggest that Bcl-2 may protect cell from TGF--F-induced apoptosis by interfering TGF- generated signals leading to induce reactive oxygen species production.     

Phosphorus-31 nuclear magnetic resonance study on the effects of endurance training in rat skeletal muscle

To evaluate changes in muscle energetics following endurance training, we measured phosphorus-31 nuclear magnetic resonance (31P NMR) spectra on rat muscle in vivo before and after training in the same animals. The endurance training lasted for 3 months. The 31P NMR spectra were obtained serially at rest, during exercise by electrical stimulation, and during recovery. Intramuscular phosphocreatine (PCr), inorganic phosphate (P(i)), adenosine 5'-triphosphate (ATP) and pH were determined from the NMR spectra. The ratio of PCr:(PCR + P(i) at rest showed no difference between the trained and control groups even after 3 months of training. During exercise, however, this ratio was significantly higher in the trained group than in the control group. The ratio also recovered more rapidly after exercise in the trained group. The intramuscular pH decreased slightly by approximately 0.1 pH unit during exercise but did not show a significant difference between the groups. These results indicated that endurance training of 3 months duration improved the ATP supply system in the muscle. They also demonstrated that 31P NMR is a potent method for evaluating the effects of training in the same individuals.     

Damage and protection of the photosynthetic apparatus from UV-B radiation. I. Effect of ascorbate

In this work, the effect of the exogenously added ascorbate (Asc) against the UV-B inhibition of the photosystem II (PSII) functions in isolated pea thylakoid membranes was studied. The results reveal that Asc decreases the UV-B induced damage of the donor and the acceptor side of PSII during short treatment up to 60 min. The exogenous Asc exhibits a different UV-protective effect on PSII centers in grana and stroma lamellae, as the effect is more pronounced on the PSIIβ centers in comparison to PSIIα centers. Data also suggest that one of the possible protective roles of the Asc in photosynthetic membranes is the modification of the oxygen-evolving complex by influence on the initial S₀–S₁ state distribution in the dark.     

Fenofibrate exhibits a high potential to suppress the formation of squamous cell carcinoma in an oral-specific 4-nitroquinoline 1-oxide/arecoline mouse model

The excessive use of areca nut and/or tobacco may induce the production of free radicals and reactive oxygen species, which affect the lipid contents of the cell membrane and are possibly involved in tumorigenic processes in the oral cavity. The aim of this study was to investigate the therapeutic efficacy of fenofibrate (0.1% or 0.3%, w/w), a ligand of the peroxisome proliferator-activated receptor alpha (PPARα), in a 4-nitroquinoline 1-oxide (4-NQO)/arecoline-induced oral cancer mouse model. The carcinogen, 4-NQO/arecoline, was administrated to C57BL/6JNarl mice for 8 weeks followed by fenofibrate treatment for 12 or 20 weeks. After 28 weeks, changes in serum lipids, the multiplicity of tumor lesions, and tumor sizes were determined together with changes in the immunohistochemical expressions of PPARα, acetyl-coenzyme A carboxylase (ACC), the epidermal growth factor receptor (EGFR), and cyclooxygenase-2 (COX2). The results showed that when compared to the 4-NQO/arecoline only group, 0.3% fenofibrate treatment increased serum total cholesterol, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels. 0.3% fenofibrate treatment suppressed the incidence rate of tongue lesions, reduced the multiplicity of squamous cell carcinoma (SCC), decreased the tumor size, and increased the immunoreactivity of EGFR and COX2 in oral dysplasia but decreased EGFR and COX2 expressions in SCC. These findings indicated that fenofibrate reduced the tumor incidence rate and suppressed the tumor progression into SCC and that these molecular events might be linked to the EGFR and COX2 regulatory pathways. We suggest that fenofibrate provides a new strategy for preventing oral tumor progression.     

Glycinebetaine alleviates the inhibitory effect of moderate heat stress on the repair of photosystem II during photoinhibition

Transformation with the bacterial gene codA for choline oxidase allows Synechococcus sp. PCC 7942 cells to accumulate glycinebetaine when choline is supplemented exogenously. First, we observed two types of protective effect of glycinebetaine against heat-induced inactivation of photosystem II (PSII) in darkness; the codA transgene shifted the temperature range of inactivation of the oxygen-evolving complex from 40-52 °C (with half inactivation at 46 °C) to 46-60 °C (with half inactivation at 54 °C) and that of the photochemical reaction center from 44-55 °C (with half inactivation at 51 °C) to 52-63 °C (with half inactivation at 58 °C). However, in light, PSII was more sensitive to heat stress; when moderate heat stress, such as 40 °C, was combined with light stress, PSII was rapidly inactivated, although these stresses, when applied separately, did not inactivate either the oxygen-evolving complex or the photochemical reaction center. Further our studies demonstrated that the moderate heat stress inhibited the repair of PSII during photoinhibition at the site of synthesis de novo of the D1 protein but did not accelerate the photodamage directly. The codA transgene and, thus, the accumulation of glycinebetaine alleviated such an inhibitory effect of moderate heat stress on the repair of PSII by accelerating the synthesis of the D1 protein. We propose a hypothetical scheme for the cyanobacterial photosynthesis that moderate heat stress inhibits the translation machinery and glycinebetaine protects it against the heat-induced inactivation.     

Cellular energization protects the photosynthetic machinery against salt-induced inactivation in Synechococcus

The effects of the energization of cells by light and by exogenous glucose on the salt-induced inactivation of the photosynthetic machinery were investigated in the cyanobacterium Synechococcus sp. PCC 7942. The incubation of the cyanobacterial cells in a medium supplemented with 0.5 M NaCl induced a rapid decline with a subsequent slow decline, in the oxygen-evolving activity of Photosystem (PS) II and in the electron-transport activity of PSI. Light and exogenous glucose each protected PSII and PSI against the second phase of the NaCl-induced inactivation. The protective effects of light and glucose were eliminated by an uncoupler of phosphorylation and by lincomycin, an inhibitor of protein synthesis. Light and glucose had similar effects on the NaCl-induced inactivation of Na⁺/H⁺ antiporters. After photosynthetic and Na⁺/H⁺-antiport activities had been eliminated by the exposure of cells to 0.5 M NaCl in the darkness, both activities were partially restored by light or exogenous glucose. This recovery was prevented by lincomycin. These observations suggest that cellular energization by either photosynthesis or respiration, which is necessary for protein synthesis, is important for the recovery of the photosynthetic machinery and Na⁺/H⁺ antiporters from inactivation by a high level of NaCl.