Fe-Mn doped powdered activated carbon pellet as ozone catalyst for cost-effective phenolic wastewater treatment: Mechanism studies and phenol by-products elimination.

A novel bimetallic doped PAC (Fe-Mn/PAC) pellet was prepared with a facile sol-gel method and used as an ozone catalyst for phenolic wastewater (PWW) treatment. Adoption of Fe-Mn/PAC pellet in microbubble ozonation enhanced the 1-h chemical oxygen demand (COD) and phenol removal in PWW to 79% and 95%, respectively. With ozone dosage of 10 mg/L, 1 g/L Fe-Mn/PAC pellet exhibited ozone conversion of 92%. In comparison to microbubble ozonation process, Fe-Mn/PAC induced microbubble-catalytic ozonation process promoted ozone decomposition rate by 1.9 times. In terms of •OH production, Fe-Mn/PAC pellet enhanced •OH exposure by 10 times, with a Rct value of 2.92 × 10 -8. Rct kinetic model also suggested that Fe-Mn/PAC pellet obtained higher kinetic rate constants for initiating and promoting •OH generation. Usage of Fe-Mn/PAC pellet in microbubble ozonation for phenolic wastewater treatment also reduced the total ozone consumption by 70%. In Fe-Mn/PAC induced microbubble-catalytic ozonation process, the ratio between ozone consumption and COD removal (ΔO3/ΔCOD) was 0.91. Fe-Mn/PAC pellet characterization with X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared (FT-IR) and X-ray powder diffraction (XRD) analysis revealed successful doping of Fe-Mn on PAC substrate and larger numbers of carbon-oxygen/hydroxyl surface groups, which played key roles in ozone decomposition and •OH production.

Reverse osmosis concentrate treatment by microbubble ozonation-biological activated carbon process: Organics removal performance and environmental impact assessment.

Reverse osmosis (RO) is being used in many water reclamation facilities to produce high quality water that can be reused for different purposes. As a part of the RO process, a reject stream is produced as the reverse osmosis concentrate (ROC), which contains elevated levels of contaminants compared to the source water. Effective treatment and safe disposal of ROC via cost-effective means is very challenging. This study aims to develop a robust microbubble ozonation-biological process for industrial ROC treatment with a target effluent chemical oxygen demand (COD) lower than 60 mg/L. As compared to macrobubble ozonation, microbubble ozonation exhibited better ozone dissolution and 29% higher COD removal efficiency with the same ozone dosage. Under the optimum operating conditions with ozone dosage of 30 mg/L, ROC natural pH of 8.67 and ozonation duration of 1 h, microbubble ozonation achieved 42% COD removal efficiency while increasing the BOD5/COD ratio (ratio of biological oxygen demand over 5 days to the corresponding chemical oxygen demand) in ROC from 0.042 to 0.216. A biological activated carbon (BAC) column with an empty bed contact time (EBCT) of 120 min was combined with microbubble ozonation for continuous ROC treatment. Over the 100-day operation, the combined system performed consistent organics removal with an average effluent COD of 45 mg/L. Both LC-OCD data and fluorescence EEM spectra confirmed humic substances were the dominant organic species in ROC. Ozone pre-treatment could achieve significant removal of humic substances in raw ROC. ATP analysis found that ozone pre-treatment enhanced BAC biofilm activity by around 5 folds. 5 min acute toxicity assessment with Aliivibrio fischeri showed 4 times reduction of bioluminescence inhibition in ozone treated ROC. From the environmental point of view, Life cycle assessment (LCA) results demonstrated that Ozone-BAC system had significant environmental burdens on climate change and human toxicity due to the electricity production process. These environmental impacts can be mitigated by optimizing the ozonation process with reduced ozone dosage or utilizing renewable energy sources for electricity generation.

Simulation of FBR-Fenton/GAC process for recalcitrant industrial wastewater treatment with a computational fluid dynamics-kinetic model framework.

An integrated computational fluid dynamics (CFD)-kinetic model framework was developed to numerically describe the hydrodynamic and kinetic phenomena in a liquid-solid two phases Fluidized-bed reactor Fenton/granular activated carbon (FBR-Fenton/GAC) system. The model obtained excellent accuracy for predicting chemical oxygen demand (COD) removal in reverse osmosis concentrate (ROC) treatment under different operation conditions. Hydrodynamic evaluation demonstrated that under the quasi-steady state, the GAC particles were uniformly circulated in the bed region with two pairs of counter-rotating recirculation cells, and a clear interface layer formed between the solid and the liquid phases. Superficial liquid velocity highly affected the fluidized bed expansion and solid volume fraction, while its impact on the overall COD removal efficiency was negligible. Chemical evaluation revealed that GAC/H2O2 catalytic reaction enhanced the •OH production in FBR-Fenton/GAC process by 2.7 folds as compared to homogenous Fenton process. Fenton reaction mainly occurred in the upper liquid region and its kinetics for •OH generation significantly diminished by 75% within the first 10 min. GAC/H2O2 reaction took place in the fluidized bed region for continuous •OH generation with a relatively stable rate from 1.21 × 10-6 to 0.60 × 10-6 M/s. Along the ROC treatment with FBR-Fenton/GAC process, the simulated COD degradation rate decreased along the reaction time with 2.05 × 10-6 M/s and 2.93 × 10-7 M/s at 2 min and 60 min, respectively. Faster COD removal was attained in the fluidized bed region due to combining effects of •OH oxidation and GAC adsorption. The overall predicted COD concentration reduced from 122 to 35 mg/L, •OH oxidation and GAC adsorption contributed 59% and 41%, respectively, to the total COD removal.

Organics removal in high strength petrochemical wastewater with combined microbubble-catalytic ozonation process.

Ozonation is a well-known and widely applied advanced oxidation process (AOP) for industrial wastewater treatment, while the ozonation efficiency might be limited by low mass transfer, poor solubility, and rapid decomposition rate of ozone molecules in the aqueous phase. The present study aims to investigate the feasibility of combined microbubble-catalytic ozonation process (M-O3/Fe/GAC) for improving the ozonation efficiency during treatment of petrochemical wastewater (PCW). M-O3/Fe/GAC process optimization was carried out with different pH conditions, ozone dosages and catalyst loadings. The optimum operating conditions were identified as 50 mg L-1 ozone dosage, real PCW pH (7.0-7.5) and 4 g L-1 catalyst loading. Among different ozonation processes, M-O3/Fe/GAC process achieved the highest chemical oxidation demand (COD) removal efficiency of 88%, which is 18% and 43% higher than those achieved by the microbubble and macrobubble ozonation processes, respectively. Phenolic compounds presented in PCW could be reduced by 63% within 15 min in M-O3/Fe/GAC treatment process. Long-term continuous flow studies suggested M-O3/Fe/GAC process to be the most cost-effective technology for PCW treatment with an operating cost of S$0.18 kg-1 COD and S$0.4 m-3 with good catalyst stability. Liquid size exclusion chromatography with organic carbon detection (LC-OCD) data suggested humic substances to be the dominant organic species in PCW, M-O3/Fe/GAC could achieve significant humic substances removal and biodegradability enhancement in PCW. Kinetics and mechanism studies revealed that organics removal in M-O3/Fe/GAC was 1.8 times higher than that in microbubble ozonation process, and hydroxyl radical (●OH) was the dominant oxidant specie for organics removal in M-O3/Fe/GAC process.

Fluidized-bed Fenton technologies for recalcitrant industrial wastewater treatment-Recent advances, challenges and perspective.

In recent years, fluidized-bed Fenton (FBR-Fenton) process has gained more attention in treating recalcitrant industrial wastewater. FBR-Fenton combines the effectiveness of homogeneous Fenton and sludge reduction of heterogeneous Fenton. Comparing to other modified Fenton processes, FBR-Fenton has greater economical and scaling up potential. However, large consumption of Fenton reagents and strict pH control are still the bottlenecks hampering the full-scale application of FBR-Fenton. While prior reviews mainly focused on the operation and performance of FBR-Fenton process, the present study critically discussed the challenges and bottlenecks for its full-scale industrial application. This study also comprehensively reviewed the development strategies for tackling these drawbacks, mainly over the recent five years. Homogeneous FBR-Fenton, heterogeneous FBR-Fenton and heterogeneous FBR-photo-Fenton processes were classified for the first time according to their reaction mechanisms and system designs. Important operational and design parameters affecting the cost-effectiveness of all FBR-Fenton technologies were reviewed, including the fundamentals, common practices and even innovative steps for enhancing the process performance. Up-to-date applications of FBR-Fenton technologies in recalcitrant wastewater/compounds treatment were also summarized, and it was found that upscaling of heterogeneous FBR-Fenton and heterogeneous FBR-photo-Fenton processes was still very challenging. Strategies to overcome the key technical limitations and enhance process cost-effectiveness were discussed in the future perspective part. Furthermore, modelling techniques such as computational fluid dynamics model and artificial neural network were suggested to be promising modelling techniques for speeding up the full-scale applications of FBR-Fenton technologies.

Organics removal and in-situ granule activated carbon regeneration in FBR-Fenton/GAC process for reverse osmosis concentrate treatment.

Fluidized bed reactor Fenton (FBR-Fenton) process was adopted for reverse osmosis concentrate (ROC) treatment with three types of carriers, including sand, zeolite and granular activated carbon (GAC). Adsorption studies demonstrated that GAC achieved the best adsorption performance (maximum COD removal of 78% in 15 h) among the three carriers, and the adsorption of ROC organic matters followed a two-stage adsorption model. Fenton oxidations were carried out in three fluidized beds after column saturation, and FBR-Fenton/GAC process achieved highest COD removal (72%) and most BOD5/COD ratio enhancement (from 0.03 to 0.3) in ROC. Long-term operation data demonstrated good performance stability of GAC as the carrier. In addition, GAC fluidized bed obtained highest total iron removal rate via iron crystallization process. Continuous in-situ GAC regeneration with more than 90% recoveries of surface area, pore volume and adsorption capacity were observed along the ROC treatment with FBR-Fenton/GAC process. Mechanism studies revealed that better COD removal performance in FBR-Fenton/GAC process was attributed to the combining effects of homogenous Fenton reaction, GAC adsorption and GAC/H2O2 catalytic reaction.

Proteomic alterations of fibroblasts induced by ovarian cancer cells reveal potential cancer targets.

The common spread pattern of ovarian cancer is peritoneal implantation. The growth of the shed ovarian cancer cells in the peritoneal cavity is closely related to the tumor microenvironment. Cancer-associated fibroblasts are vital in the tumor microenvironment. It is not clearly defined that the protein expression alters during the activating process of fibroblasts. This study detected the protein alterations in fibroblasts induced by ovarian cancer cells and explored the potential biological relevance through two-dimensional gel electrophoresis and mass spectrometry. Our data showed that the level of CENPE, BAG2, SOD2, GDI2, CORO1C, CFL1, DSTN, CALD1, PHGDH, PDHA1, AKR1B1, TST and TBCA proteins were significantly up-regulated in the fibroblasts co-cultured with ovarian cancer cells, whereas HSPB1, P4HB and VIM were significantlydown-regulated. However, only BAG2, SOD2 and CORO1C proteins were confirmed to be significantly increased by western blot analysis. The differentially expressed proteins were mainly involved in metabolic processes, cellular component organization, responses to stimulus, multicellular organismal processes, localization, protein depolymerization, cellular senescence and the mitotic pathway. These data demonstrated that fibroblasts had an altered protein expression pattern after being induced by ovarian cancer cells, and participated in multiple cell processes resulting in tumor progression. The differentially expressed proteins should be considered as targets for cancer treatment.

[Intervention of edaravone against renal injury induced by acute paraquat poisoning in rats].

Objective: To investigate the dynamic change of paraquant-induced kidney injury in rats and the protective effect of edaravone. Methods: Eighty SD rats were randomly divided into 4 groups: the normal control group, paraquat poisoning group, edaravone treatment group and edaravone control group. The normal control group of 8 rats were given 1 ml of 0.9% sodium chloride through the abdominal cavity, and the same amount of fluid into the abdominal cavity after 30 minutes. The paraquat poisoning group of 24 rats were given 1 ml of paraquat solution (20 mg/kg) through the abdominal cavity to build poisoning models, and the same amount of 0.9% sodium chloride was injected into the abdominal cavity after 30 minutes. The edaravone treatment group of 24 rats were given edaravone (5 mg/kg) through the abdominal cavity after 30 minutes when the poisoning models were set up. The edaravone control group of 24 rats were given 1 ml of 0.9% sodium chloride through the abdominal cavity, and edaravone (5 mg/kg) was injected into the abdominal cavity after 30 minutes. In addition to the normal control group, the other groups processed 1 times a day to mantain 7 d. On 1, 3, 7, 21 d several rats in each group were excuted and the kidney tissue and serum samples were collected, then each pathological changes of the kidney were observed with light microscopy. Serum creatinine, KIM-1, NGAL were measured by ELISA, the expression of HSP70 protein in kidney were observed with immunohistochemical staining. Results: The pathological examination reveald that the damage of kidney tissue in the paraquat group was the most serious on 3 d, and the damage was consistently alleviated in edaravone treatment group at the same time, renal fibrosisn was unseen in each group until 21 d. Compared with normal control group, there was no statistically significant in edaravone control group (P>0.05) . The KIM-1 in blood and kidney in paraquat poisoning group were markedly increased in 1 d (P<0.05) . The NGAL in blood and creatinine were markedly increased in d7 (P<0.05) . The NGAL in kidney increased over time, but had no statistically difference with the control group (P>0.05) .Compared with paraquat poisoning group, the serum creatinine, KIM-1 in blood and kidney, the KIM-1 in kidney had decreased significantly in edaravone treatment group (P<0.05) . The NGAL in kidney has no statistically significant compared with the poisoning group (P>0.05) . HSP70 expression of kidney tissue in edaravone treatment group had significantly increased in d3 compared with the paraquat poisoning group (P<0.05) . Conclusion: Edaravone can prompt a significant rise of HSP70 in kidney tissue, reduce KIM-1 and NGAL levels, and play a protective role in kidney injury of acute paraquat poisoning.

[Anti-ß(2)GPâ antibodies accelerate the formation of atherosclerosis in high fat diet fed ApoE deficient mice].

Objective: To investigate the effects of anti-ß(2) glycoprotein Ⅰ(ß(2)GPⅠ) antibody on atherosclerosis in ApoE deficient mice. Methods: A total of 24 male ApoE deficient mice of specific pathogen free level(six to eight-week old)were divided into normal control group, high fat diet group, high fat diet with anti-ß(2)GPⅠ group, high fat diet with homologous control antibody group (n=6 each group). During the feeding period, mice were weighed every 2 weeks and were intraperitoneally injected with anti-ß(2)GPⅠIgG (100 µg/per) and homologous control IgG (100 µg/per) according to grouping once a week. At the 16th week, the carotid arterial lipid deposition was observed by small animal magnetic resonance imaging, and blood samples were collected from internal vein of eyeball and the concentrations of TC, TG, HDL-C and LDL-C in plasma were measured after EDTA anticoagulant treatment. AI was calculated. The mice were then sacrificed and carotid arteries were removed, hematoxylin-eosin staining was used to observe the atherosclerotic lesions near the bifurcation of carotid artery and to calculate lesion size. Results: (1) The body weight of mice was significantly higher in the high fat diet group compared to other 3 groups(all P<0.05), which was similar among high fat diet+ anti-ß(2)GPⅠantibody group, high fat diet+ homologous control IgG group and normal diet control group (P>0.05). (2) After 16 weeks, plasma concentrations of TC and LDL-C in high fat diet group, high fat diet+ anti-ß(2)GPⅠantibody group and high fat diet+ homologous control IgG group were significantly higher than in normal diet group (all P<0.05), there was no significant difference among high fat diet groups. The level of HDL-C was significantly higher in high fat diet control group than in normal diet control group. The concentration of TG was similar among groups. However, the value of AI in high fat+ anti-ß(2)GPⅠ antibody group was significantly higher than in other groups (all P<0.05). (3) After 16 weeks, magnetic resonance imaging revealed that mice in high fat diet+ anti-ß(2)GPⅠ antibody group had more obviously lipid deposition in the carotid arteries, it was significantly higher than that in the other groups, and the cross sections of carotid arteries stained with HE also demonstrated obviously carotid lumen stenosis and the percentage of carotid plaque area to carotid artery was (37.545±1.351)% in the high fat diet+ anti-ß(2)GPⅠ antibody group, it was significantly higher than normal diet group ((1.235±0.460)%), high fat diet control group((11.635±2.751)%) and high fat diet+ homologous control IgG group ((11.815±2.623)%), all P<0.01. In high fat diet+ anti-ß(2)GPⅠ antibody group, the area of carotid plaque was (3.121±0.124)×10(4) µm(2,) it was also significantly higher than normal diet group ((0.094±0.015)×10(4) µm(2)), high fat diet control group ((1.309±0.147)×10(4) µm(2)) and high fat diet+ homologous control IgG group ((1.027±0.228)×10(4)µm(2)), all P<0.01. Conclusion: Anti-ß(2)GPⅠ antibody can promote atherosclerotic plaque formation in high fat diet fed ApoE deficient mice.

Prognostic study for overall survival in patients with newly diagnosed POEMS syndrome.

POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes) is a multisystem disorder with a good long-term prognosis. In its dozens of clinical features, those with independent prognostic value are still not well characterized. We retrospectively included 362 patients with newly diagnosed POEMS syndrome at our institute from 2000 to 2015. On the basis of a randomized sample splitting, we first identified four baseline clinical variables, including age >50 years (hazards ratio (HR) 4.07, 95% confidence interval (CI) 1.41-11.76, P=0.009), pulmonary hypertension (HR 3.99, 95% CI 1.44-11.04, P=0.008), pleural effusion (HR 3.81, 95% CI 1.23-11.79, P=0.02) and estimated glomerular filtration rate <30 ml/min/1.73 m2 (HR 8.25, 95% CI 2.18-31.25, P=0.002), associated with inferior overall survival in the derivation cohort, with the use of multivariate Cox regression model. These factors were incorporated together to develop a prognostic nomogram. Concordance index calculation (0.727, 95% CI 0.601-0.853, P=0.018) and calibration curve plotting demonstrated its significant predictive and discriminatory capacity in the validation cohort. This nomogram could be a useful and convenient tool in clinical practice to evaluate individualized prognosis in patients with newly diagnosed POEMS syndrome.

Mifepristone-inducible recombinant adenovirus attenuates paraquat-induced lung injury in rats.

OBJECTIVE: To investigate the effects of overexpression of nuclear factor E2-related factor-2 (NRF2) on lung injury in rats exposed to paraquat (PQ) poisoning. METHODS: A mifepristone (RU486)-inducible recombinant adenoviral vector carrying the human NRF2 gene (Ad-RUNRF2) was constructed and transfected via airway into the rats 7 days before the administration of RU486. Rats were orally challenged with PQ at 20 mg/kg 24 h after the injection of RU486. On days 0.5, 3 and 21 after PQ poisoning, the expressions of NRF2 and cytokines related to inflammation and oxidation in lung tissue were examined. RESULTS: RU486 remarkably enhanced NRF2 mRNA and NRF2 protein levels in Ad-RUNRF2-transfected rats in a dose-dependent manner (p < 0.01). PQ stimulated compensatory overexpression of NRF2, heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO-1) in lungs on days 0.5 and 3 after exposure (p < 0.05), but depleted the expression of catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione (GSH), with an increased malondialdehyde (MDA) (p < 0.05). However, pretreatment with Ad-RUNRF2 and RU486 strongly enhanced the expression levels of NRF2, HO-1, NQO-1, CAT and GSH-Px in the lungs of PQ intoxicated rats, with increased GSH and decreased MDA (p < 0.05). Pretreatment with Ad-RUNRF2 and RU486 also strongly suppressed the PQ-induced activation of nuclear factor κB (NF-κB) and decreased the levels of tumour necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). In addition, Ad-RUNRF2 and RU486 induction significantly reduced PQ-induced pathological changes in lungs and attenuated lung oedema and protein leakage caused by PQ (p < 0.05). CONCLUSION: RU486-induced overexpression of NRF2 in lungs transfected with Ad-RUNRF2 can ameliorate PQ-induced lung injury by the activation of the NRF2-antioxidant response element (ARE) pathway.

Primary lymphoepithelioma-like carcinoma of ocular adnexa: clinicopathologic features and treatment.

BACKGROUND AND METHODS: Lymphoepithelioma-like carcinoma (lelc) is a rare malignancy in ocular adnexa. Here, we report 4 patients with lelc and review 11 patients reported in the literature. Clinical profiles, association with Epstein-Barr virus (ebv), treatment, and outcomes are analyzed. RESULTS: Lacrimal glands and the lacrimal drainage system, eyelid, and conjunctiva are potential primary sites for lelc. The tumours are characterized histologically by nests of undifferentiated malignant cells surrounded by lymphoid infiltrates. Infection with ebv was confirmed in lelc of ocular adnexa, and that association seemed to be restricted to Asian populations. Results from our centre uniformly showed expression of ebv-encoded small rnas in primary tumour, locally recurrent tumour, and metastatic lymph nodes. This disease had a tendency to relapse regionally. Postoperative radiotherapy seems to improve disease-free survival. Tumours appear to be sensitive to radiotherapy and chemotherapy based on cisplatin and 5-fluorouracil. At our centre, 3 patients were still living at 22, 33, and 76 months after surgery. One patient died of distant metastasis after a survival of 38 months. CONCLUSIONS: Lymphoepithelioma-like carcinoma is a heterogenous entity among ocular adnexal malignancies. Multimodality treatment provides a better chance at survival. Further investigation is required to achieve a better understanding of the biologic behavior of this entity and of its optimal treatment.

The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation.

We have identified a basic sequence in the N-terminal region of the 67-kDa serum response factor (p67SRF or SRF) responsible for its nuclear localization. A peptide containing this nuclear localization signal (NLS) translocates rabbit immunoglobulin G (IgG) into the nucleus as efficiently as a peptide encoding the simian virus 40 NLS. This effect is abolished by substituting any two of the four basic residues in this NLS. Overexpression of a modified form of SRF in which these basic residues have been mutated confirms the absolute requirement for this sequence, and not the other basic amino acid sequences adjacent to it, in the nuclear localization of SRF. Since this NLS is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase), we further investigated if A-kinase plays a role in the nuclear location of SRF. The nuclear transport of SRF proteins requires basal A-kinase activity, since inhibition of A-kinase by using either the specific inhibitory peptide PKIm or type II regulatory subunits (RII) completely prevents the nuclear localization of plasmid-expressed tagged SRF or an SRF-NLS-IgG conjugate. Direct phosphorylation of SRF by A-kinase can be discounted in this effect, since mutation of the putative phosphorylation sites in either the NLS peptide or the encoded full-length SRF protein had no effect on nuclear transport of the mutants. Finally, in support of an implication of A-kinase-dependent phosphorylation in a more general mechanism affecting nuclear import, we show that the nuclear transport of a simian virus 40-NLS-conjugated IgG or purified cyclin A protein is also blocked by inhibition of A-kinase, even though neither contains any potential sites for phosphorylation by A-kinase or can be phosphorylated by A-kinase in vitro.

17 beta Hydroxysteroid dehydrogenase 1 "pseudogene" is differentially transcribed: still a candidate for the breast-ovarian cancer susceptibility gene (BRCA1).

BRCA1, the susceptibility gene for hereditary breast-ovarian cancer, is located on chromosome 17q12-21 but has not yet been identified. Two tandem oestradiol 17 beta hydroxysteroid dehydrogenase genes (17HSD) are assigned to this region. The active 17HSDII gene encodes the normal enzyme which regulates local synthesis of oestrogens, whereas 17HSDI is considered to be a pseudogene. We used reverse transcription coupled to polymerase chain reaction (RT-PCR) and found that the 17HSDI gene was also transcribed in half of the human cell lines and most of the biopsies studied, suggesting that 17HSDI could modulate normal 17HSDII activity in oestrogen target cells. We hypothesize that altered 17HSDI gene expression could lead to both hereditary and/or sporadic breast cancer by increasing local oestrogen concentration and that it is still a potential candidate for BRCA1.

Chromosomal location and expression of the genes coding for Ku p70 and p80 in human cell lines and normal tissues.

Ku protein is a relatively abundant DNA-binding nuclear protein complex composed of two polypeptide subunits, p70 and p80. Ku has been recently identified as the regulatory component of the DNA-dependent protein kinase that phosphorylates RNA polymerase II. To further characterize in vivo regulation of Ku protein, we studied the expression of the transcripts coding for the Ku p70 and p80 subunits in different human cell lines and normal tissues by Northern blot hybridization, using specific cDNA probes. The expression level of both genes was approximately 10-fold higher in established cell lines than in normal tissues. However, mRNA expression levels in permanent cell lines correlated more strongly with their proliferative state than with their level of malignant transformation. In purified T lymphocytes induced to proliferate by the combined action of monoclonal antibodies directed against the CD2 and CD28 adhesion molecules, Ku p70 and p80 mRNA steady-state levels increased as soon as 6 h after activation and lasted at least 72 h. The human genes coding for the Ku p70 and p80 subunits were localized by cytogenetic mapping, using fluorescence in situ hybridization, to 22q13 and 2q33-->q35, respectively.

Expression and purification of the DNA-binding domain of SRF: SRF-DB, a part of a DNA-binding protein which can act as a dominant negative mutant in vivo.

We have developed an approach which allows functional in vivo examination of DNA-binding proteins through microinjection of polypeptides containing the DNA-binding domain into living fibroblasts. The present analysis utilizes serum response factor (SRF), a transcription factor that binds to the serum response element. We have expressed in bacteria a 30-kDa portion of this protein (amino acids 113 to 265) containing the DNA-binding domain of SRF (SRF-DB) and purified it to homogeneity by a single DNA affinity chromatography step using the high-affinity SRF-binding site (ACT.L). We have tested the efficiency of SRF-DB to prevent endogenous SRF function through analysis of c-fos expression and DNA synthesis stimulated by fetal calf serum, two events known to require SRF. Injection of purified SRF-DB into rat embryo fibroblasts inhibits c-fos induction by growth factors. Moreover, DNA synthesis, induced after serum addition, is also suppressed by SRF-DB injection. This implies that overproduction of SRF-DB makes the cell deficient in the function of wild-type SRF and that SRF-DB acts as a dominant negative mutant. These data show that, for the study of DNA-binding proteins, expressing and using portions of the protein that corresponds to the DNA-binding domain present a useful method for generating dominant negative mutants and illustrate the potential application of the DNA-binding region to facilitate the study of events at the DNA/protein level.