[Oxidative damage related to PM2.5 exposure in human embryonic stem cell-derived fibroblasts].

OBJECTIVE: To study the oxidative damage of PM2.5 in human embryonic stem cells (EBf-H9 cells), and to provide a theoretical basis for revealing the adverse health effects of PM2.5 and the potential mechanisms, and also to provide a new alternative cell model for PM2.5 risk assessment. METHODS: EBf-H9 cells were cultured with 0.00 (the constrast group) 3.91, 7.81, 15.63, 31.25, 62.50, 125.00 µg/cm(2) of PM2.5. CCK-8 kit was used to determine the survival rate of cells exposed to PM2.5 for 6 h. DCFH-DA probe was used to detect the total ROS content of cells exposed to PM2.5 for 1 h. The activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px), and the content of lipid peroxides such as malondialdehyde (MDA) in cells exposed to PM2.5 for 6 h were detected by using the commercial kits. ANOVA model analyzed the statistical significance from the different concentration group. RESULTS: The cytotoxicity results showed that the cell survival rate was decreased gradually with the increase of the concentrations of PM2.5, and the half inhibitory concentration (IC50) was 83.01 µg/cm(2). When the exposure concentration was 3.91, 7.81, 15.63, 31.25, 62.5 µg/cm(2), after exposure of PM2.5 1 h, the ROS florescence was 27.12±0.21, 54.03±0.50, 60.93±0.08, 61.36±1.00, 68.21±0.93, 78.27±1.26 (compared to control group 27.12±0.21, all P level<0.01). After exposure of PM2.5 6 h, the activities of T-SOD was (9.78±0.28), (8.59±0.22), (8.90±0.33), (7.46±0.71), (4.21±0.17) U/mg protein (F=98.881, compared to control group (11.77±0.63) U/mg protein, all P level<0.01). The activities of GSH-Px was (181.59±3.65), (153.33±1.69), (168.74±2.22), (81.56±0.56), (48.62±2.13) U/mg protein (compared to control group (273.90±6.50) mU/mg protein, all P level<0.01). And the content of MDA was (0.38±0.03), (0.43±0.09), (0.47±0.09), (0.65±0.10), (0.70±0.12) nmol/mg protein (compared to control group (0.27±0.02) nmol/mg protein, all P level<0.05). CONCLUSION: PM2.5 exposure can decrease EBf-H9 cells viability, and improve the levels of lipid peroxidation. It may be due to induce EBf-H9 cells to increase the production of ROS and to make the cells appear oxidative stress, which lead to oxidative damage to cells. The present study reveals the mode of action of PM2.5 in terms of oxidative damage to EBf-H9 cells. It is also indicated that the cells may be a new alternative cell model for PM2.5 risk assessment.

[PM2.5 induces oxidative damage and affects nuclear factor-erythroid 2 related factor 2 pathway in human umbilical vein endothelial cells].

OBJECTIVE: To assess the oxidative damage after exposure to fine particulate matter (PM2.5) in human umbilical vein endothelial cells (HUVECs) and to explore the influence of the Nrf2 pathway. METHODS: HUVECS were exposed to different concentrations of PM2.5 as follows: 0.000 (control), 0.004, 0.039, 0.391, 1.950, 3.910, 7.810, 15.600, 31.250, 62.500, 125.000 and 250.000 µg/cm(2). After 24 h, cell viability was measured by the CCK-8 method. In a separate experiment, HUVECs were exposed to 0 (control), 1.95, 3.91, 7.81, 15.63 or 31.25 µg/cm(2) of PM2.5. The level of cellular reactive oxygen species (ROS) was detected with an H2-DCFDA fluorescence probe after 1h and 3 h exposure. After 24 h exposure, the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx), and malondialdehyde (MDA) content were detected by colorimetry. Western blot was used to estimate the expression levels of Nrf2 and NQO1 in total protein. RESULTS: HUVEC viability was reduced in a concentration-dependent manner by PM2.5. Compared with controls (100% viability), the viability of the 250 µg/cm(2) group was (38.18±6.68)% (P<0.05). Substantial accumulation of ROS occurred in HUVEC after 1 h and 3 h exposure to PM2.5. After 24 h exposure to 0, 1.95, 3.91, 7.81, 15.63 and 31.25 µg/cm(2) of PM2.5, SOD activity decreased concentration-dependently to (26.25±1.76), (24.99±1.81), (24.25±0.49), (22.07±1.13), (21.03±0.43) and (19.37±0.84) U/mg protein, respectively, (F=13.95, P<0.001). GPx activity decreased in a concentration-dependent manner to (25.63±1.33), (24.40±2.20), (22.85±2.46), (20.98±1.95), (20.17±1.86) and (18.69±3.11) mU/mg protein, respectively (F=4.26, P=0.019), whereas MDA increased concentration-dependently to (1.11±0.07), (1.12±0.07), (1.17±0.05), (1.49±0.01), (1.95±0.08) and (2.37±0.08) nmol/mg protein, respectively, (F=186.37, P<0.001). Compared with the control Nrf2, NQO1 protein levels (1.00±0.27, 1.00±0.33), 15.63 µg/cm(2) group (2.38±0.44, 1.78±0.20) were enhanced (P<0.05). CONCLUSION: These results demonstrate that PM2.5 can lead to oxidative damage to HUVEC in a concentration-dependent manner. Protein levels of Nrf2 and NQO1 were enhanced at high concentrations of PM2.5, and this mechanism may be related to increases in cellular ROS induced by PM2.5.

Prepubertal exposure to T-2 toxin advances pubertal onset and development in female rats via promoting the onset of hypothalamic-pituitary-gonadal axis function.

T-2 toxin, a naturally produced Type A trichothecene mycotoxin, has been shown to damage the reproductive and developmental functions in livestocks. However, whether T-2 toxin can disturb the pubertal onset and development following prepubertal exposure remains unclear. To clarify this point, infantile female Sprague-Dawley rats were given a daily intragastric administration of vehicle or T-2 toxin at a dose of 375 µg/kg body weight for 5 consecutive days from postnatal day (PND) 15-19 (PND15-PND19). The days of vaginal opening, first diestrus, and first estrus in regular estrous cycle were advanced following T-2 toxin treatment, indicating prepubertal exposure to T-2 toxin induced the advancement of puberty onset. The relative weights of uterus and ovaries and the incidence of corpora lutea were all increased in T-2 toxin-treated rats; serum hormone levels of luteinizing hormone and estradiol and the messenger RNA expressions of gonadotropin-releasing hormone (GnRH) and GnRH receptor also displayed marked increases following exposure to T-2 toxin, all of which were well consistent with the manifestations of the advanced puberty onset. In conclusion, the present study reveals that prepubertal exposure to a high level of T-2 toxin promotes puberty onset in infantile female rats by advancing the initiation of hypothalamic-pituitary-gonadal axis function in female rats.

Cassava (Manihot esculenta Krantz) genome harbors KNOX genes differentially expressed during storage root development.

In plants, homeodomain proteins play a critical role in regulating various aspects of plant growth and development. KNOX proteins are members of the homeodomain protein family. The KNOX transcription factors have been reported from Arabidopsis, rice, and other higher plants. The recent publication of the draft genome sequence of cassava (Manihot esculenta Krantz) has allowed a genome-wide search for M. esculenta KNOX (MeKNOX) transcription factors and the comparison of these positively identified proteins with their homologs in model plants. In the present study, we identified 12 MeKNOX genes in the cassava genome and grouped them into two distinct subfamilies based on their domain composition and phylogenetic analysis. Furthermore, semi-quantitative reverse transcription polymerase chain reaction analysis was performed to elucidate the expression profiles of these genes in different tissues and during various stages of root development. The analysis of MeKNOX expression profiles of indicated that 12 MeKNOX genes display differential expressions either in their transcript abundance or expression patterns.

Cloning and molecular characterization of a cDNA encoding a small GTPase from Hevea brasiliensis.

Small GTPases play a critical role in the regulation of a range of cellular processes including growth, differentiation, and intracellular transportation. The cDNA encoding a small GTPase, designated as HbGTPase1, was isolated from Hevea brasiliensis. HbGTPase1 was 882 bp long containing a 612-bp open reading frame encoding a putative protein of 203 amino acids, flanked by an 83-bp 5'-untranslated region (UTR) and a 187-bp 3'-UTR. The predicted molecular mass of HbGTPase1 is 22.62 kDa, with an isoelectric point of 5.06. The HbGTPase1 protein was predicted to possess the conserved functional regions of the small GTPase superfamily of proteins. Quantitative polymerase chain reaction analysis revealed that HbGTPase1 was constitutively expressed in all tissues tested. HbGTPase1 transcripts accumulated at relatively low levels in the flower, latex, and leaves, while HbGTPase1 transcripts accumulated at relatively high levels in bark. Transcription of HbGTPase1 in the latex was induced by jasmonate.

Constitutive expression of human angiostatin in Pichia pastoris by high-density cell culture.

A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol-0.8% PTM4 to the growing culture for 60 h at 30 degrees C. Dissolved oxygen level was maintained at 25-30% and pH was controlled at 5 by the addition of 7 M NH4OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16 melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale expression of angiostatin and other heterologous proteins.

Application of high-performance liquid chromatography-mass spectrometry to detection of diuretics in human urine.

A rapid, sensitive and reliable high-performance liquid chromatographic-mass spectrometric method for the detection of 25 diuretics in human urine has been developed. Atmosphere pressure chemical ionization (APCI) and electrospray ionization (ESI) modes were evaluated. A 2-ml volume of urine was extracted under basic conditions and separated on an Agilent Zorbax SB-C(18) column (150 x 2.1 mm, 5 microm). The mobile phase consisted of formic ammonium-formic acid buffer (pH 3.5) and acetonitrile. The effects of capillary temperature, sheath gas pressure and compositions of mobile phase on the sensitivity were studied. The recoveries of most of the diuretics were 75-95%. In the full scan mode, the limits of detection of the 25 diuretics were 0.25-25 ng/ml for APCI and 0.6-250 ng/ml for ESI. Under the optimal conditions, 14 diuretics from authentic urine samples were detected successfully by LC-APCI-MS. To obtain more fragmentation information on the chemical structure for positive confirmation, tandem mass analysis was also investigated.

[Inhibition of platelet function by L-arginine.L-aspartate salt].

The effects of L-arginine.L-aspartate salt (DR) on platelet aggregation, adhesion and release were investigated. Platelet aggregation induced by adenosine 5 -diphosphate (ADP) was significantly inhibited (P<0.01) by intravenous injection of DR (15 mg/kg) in rats or oral administration (15 mg/kg) in rabbits, the inhibitory effect on rabbit platelet aggregation lasting for more than 8 h (P<0.01). Platelet aggregation induced by ADP, collagen or thrombin in rats was all markedly inhibited by 7.5, 15 or 30 mg/kg of DR (bid for 3.5 d, ig, P<0.01). Platelet adhesion to foreign objects was inhibited by 30 mg/kg of DR (ig). Bleeding time in rat tails was prolonged by 30 mg/kg of DR (P<0.05). Furthermore, PGI(2) released from the vascular wall was increased in DR-treated rats (P<0.05), however, TXA(2) released from platelets was not affected. These data demonstrate the inhibitory effect of DR on platelet function, suggesting that its action target may be different from that of acetylsalicylic acid, and that the increase of PGI(2) release may be responsible partly for this effect. It is suggested that DR may probably be used as a new agent for regulating platelet function.

Molecular mechanism of apoptosis induced by ricin in HeLa cells.

AIM: To study the morphological changes and molecular mechanism of HeLa cell apoptosis induced by ricin. METHODS: HeLa cells were coincubated with ricin 0.05 mumol.L-1 for 1, 2, 3, 6, 12, 18, and 24 h, then scanning electron microscopy (SEM), transmission electron microscopy (TEM), Western blot cell cycle, cell cytotoxicity, and cell viability were assayed. RESULTS: The typical apoptosis was induced by ricin 0.05 mumol.L-1 and necrotic cells increased after being cultured with ricin 0.05 mumol.L-1 for more than 12 h. The apoptotic cells mainly showed cytoplasmic membrane blebbing, chromatin condensation and fragmentation, and crescentic nuclear and membrane bound apoptotic bodies formation. No detectable levels of p53, Bax, Bcl-2 and the subunit p20 of interleukin-1 beta-converting enzyme (ICE) were found by Western blot, but the active subunit p17 of 32-kDa putative cysteine protease (CPP32) was detected at 3, 6, and 9 h after ricin treatment. The activity of CPP32 in HeLa cells increased 4 to 5 folds after being treated with ricin 0.05 mumol.L-1 and reached the peak at 6 h of treatment. There was no significant difference of ICE activity between the ricin treated cells and control cells. The percentage of G2/M cells increased from 13.9% +/- 0.5% to 33.2% +/- 0.5% after 24 h of ricin 0.05 mumol.L-1 treatment. CONCLUSION: CPP32 but not ICE was involved in the ricin-induced apoptosis in HeLa cells. Ricin 0.05 mumol.L-1 had no effect on the G0/G1 phase of cell cycle, but induced G2/M arrest.

[Studies on the synthesis and activities of RGD related peptides].

In the binding of Fgn to GP IIb/IIIa, RGD is the key sequence. In the present paper, RGDS, RGDV and RGDF were synthesized by use of solution method. Bioassay indicated that the C-terminal amino acid residues were very important for their antithrombosis effects. Conformational studies showed that their antithrombosis potency may depend on their total energies. The observation of their vasodilation effects suggests that this kind of function is worthy to be further studied.

Effects of Arg-Gly-Asp-Ser on Ca2+ transport of myocardial sarcoplasmic reticulum in rat septic shock.

AIM: To study the effects of Arg-Gly-Asp-Ser (RGDS), a synthetic short peptide of fibrinogen degradation, on the Ca2+ transport function of cardiac sarcoplasmic reticulum in rat septic shock. METHODS: RGDS 5 mumol.kg-1 was injected i.v. at 4 h and 14 h after cecal ligation and puncture (CLP) operation on rats. Highly purified membrane of sarcoplasmic reticulum (SR) was prepared from rat hearts. Assays were made of ATP-dependent Ca2+ uptake by cardiac SR and [3H] ryanodine binding to SR. RESULTS: The initial rate and the capacity of SR Ca2+ uptake were increased by 104% (P < 0.01) and 12% (P < 0.05), respectively, paralleled by an increase in Ca(2+)-ATPase activity and a decrease in calcium accumulation of myo- cardium of septic rats, whereas the Bmax and Kd values of Ca2+ activated [3H]ryanodine binding to SR were unaffected after RGDS administration. CONCLUSIONS: The results indicated that RGDS have cardioprotective effects of maintaining Ca2+ homeostasis of cardiac myocytes by enhancing SR Ca2+ uptake in rat septic shock.

Synthesis and characterization of the human elastin W4 sequence.

Following the nomenclature of Sandberg, the W4 sequence of human elastin, [sequence: see text], has been synthesized by solid-phase methods and characterized by carbon-13 nuclear magnetic resonance, amino-acid analysis, mass spectra and elemental analysis. This sequence was then polymerized to greater than 50 kDa as determined by retention in 50 kDa molecular weight cut-off dialysis tubing. It has been successfully cross-linked by gamma-irradiation (20 Mrad) to form an elastomeric matrix, designated as X20-poly(W4). Physical characterizations such as stress/strain, thermolelasticity, acid-base titration and inverse temperature transition studies have been carried out on this elastomer, which is homologous to the striking, poly(VPGVG), W4 sequence of bovine and porcine elastins. These results are compared with previous results on the polypentapeptide of elastin, (VPGVG)n, and it has been demonstrated that X20-poly(W4) also is a dominantly entropic elastomer. Finally, the working model for the structure of this human elastin sequence was derived computationally using molecular mechanics and dynamics calculations. Thus the human W4 sequence appears to be structurally and functionally equivalent to the bovine and porcine W4 sequences in spite of the less regular repeating pentamer sequence.

Electro-chemical transduction in elastic protein-based polymers: a model for an energy conversion step of oxidative phosphorylation.

A pair of functional moieties, the carboxyl of an aspartic acid (Asp, D) residue and an N-methyl nicotinamide (NMeN) formed on amide linkage to the epsilon-amino group of the lysine (Lys, K) residue, are coupled to perform energy conversion by means of controlling the transition temperature, Tt, of a common hydrophobic folding and assembly domain within the polytricosapeptide, poly[GDGFP GVGVP GVGVP GFGVP GVGVP GVGK(NMeN)P]. The input of electrochemical energy in the form of the reduction of nicotinamide results in a reduction-induced increase in pKa by 2.5 pH units which represents the performance of the chemical work of picking up a proton. The primary structure and the structures of the oxidized and reduced states are verified by two-dimensional nuclear magnetic resonance. Thus electrochemical transduction, the conversion of electrochemical energy into chemical energy, has been demonstrated for the first time in a designed, synthetic protein-based polymer.

Molecular biophysics of elastin structure, function and pathology.

Owing to the presence of the recurring sequence XPGX' (where X and X' are hydrophobic residues), the molecular structure of the sequences between cross-links in elastin is viewed primarily as a series of beta-turns which become helically ordered by hydrophobic folding into beta-spirals, which in turn assemble hydrophobically into twisted filaments. Both hydrophobic folding and assembly occur when the temperature is raised above Tt, the onset of an inverse temperature transition. Using poly[fv(VPGVG),fx(VPGXG)] (where fv and fx are mole fractions with fv + fx = 1 and X is now any of the naturally occurring amino acid residues), plots of fx versus Tt result in a new hydrophobicity scale based directly on the hydrophobic folding and assembly processes of interest. With the reference values chosen at fx = 1, the most hydrophobic residues of elastin, Tyr (Y) and Phe (F), have low values of Tt, -55 and -30 degrees C, respectively, and the most hydrophilic residues, Glu (E-), Asp (D-) and Lys (K+), have high values of 250, 170 and 120 degrees C, respectively. Raising the average value of Tt for a chain or chain segment from below to above physiological temperature drives hydrophobic unfolding and disassembly; lowering Tt does the reverse. This delta Tt mechanism has been used reversibly to interconvert many energy forms and is used here to explain initiating events of elastogenesis, pulmonary emphysema, solar elastosis and the paucity of elastic fibres in scar tissue. In general, oxidation and/or photolysis convert(s) hydrophobic residues into polar residues with the consequences of irreversibly raising Tt to above 37 degrees C, hydrophobic unfolding and disassembly (fibre swelling), and greater susceptibility to proteolysis.

[The permissive action of glucocorticoid on the analgesic effect of kyotorphin and its analogue].

Kyotorphin (KTP) is an endogenous analgesic dipeptide which does not act on opiate receptors but may induce the release of endogenous opioid met-enkephalin. In order to investigate whether or not glucocorticoids have "permissive action" on KTP, hydrocortisone has been used to examine its action on the analgesic activities of KTP and its retro-isomer (riKTP) by employing thermal irradiation-tail flick method after intracerebroventricular injection. KTP and riKTP showed dose-dependent analgesic activity. Dose of KTP in the range of 6-24 mmol/L were effective, while the dose of riKTP at 6 mmol/L was shown to be ineffective. The analgesia of KTP was higher than that of riKTP. Hydrocortisone alone showed no significant analgesic effect. Linkers by connecting hydrocortisone with KTP or riKTP showed significantly higher analgesic effect compared with the corresponding dipeptides, not only in the duration of analgesia but also in potency. Solutions containing hydrocortisone and any one of the dipeptides exhibited the same effect as the linker. Pretreatment with sc naloxone (10 mg/kg) 15 min before icv of KTP or riKTP (24 mmol/L) could not block the analgesia. These findings implicate that: (1) Glucocorticoids have a permissive action on the analgesia of KTP and its retro-isomer. The glucocorticoids may exert its effects by acting on receptors in the membrane, and thus cause fast membrane effect. (2) KTP may also induce release of substances other than met-enkephalin to participate in the analgesia.

Hydrophobicity-induced pK shifts in elastin protein-based polymers.

Three polypentapeptides--poly[0.8(GVGVP), 0.2(GEGVP)], poly[0.8(GVGIP), 0.2(GEGIP)], and poly[0.75(GFGVP), 0.25(GEGVP)]--all analogues of the polypentapeptide of elastin--(Val1-Pro2-Gly3-Val4-Gly5)n or poly(VPGVG)--have been prepared to determine the effect of changing the hydrophobicity, i.e., Val1----Ile1 (I) and Val4----Phe4 (F), on the pKa and the temperature dependence of pKa of the Glu (E) residue. Shifts in pKa as large as 1.7 units are observed and the temperature dependence is much steeper for the structure-dependent proximity of the more hydrophobic Ile1 residues to the Glu4 residue. Even though this system is dominated by the inverse temperature transition of hydrophobically driven folding on raising the temperature, the effect of adding 0.15 N NaCl is to suppress the hydrophobicity-induced pKa shift.