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In this study, the East Asian diversity of green-algal Lobaria was evaluated by applying both morphological and phylogenetic approaches. A multi-locus phylogenetic analysis of 72 green-algal Lobaria specimens was performed using a three-locus and time-calibrated species-tree approach. The analyses demonstrate that pairs of sexually and vegetatively reproducing lineages split into highly supported monophyletic clades. Taxonomically, 11 green-algal Lobaria species were identified as new to science, while 10 were previously described species. The species differentiated during the Pliocene and Pleistocene. The coincidence of paleoclimatic events with estimated dates of divergence support a bioclimatic hypothesis for species evolution in the green-algal Lobaria. Molecular phylogenies, a summary of diversity, detailed new species descriptions and geographical analyses are provided. Special recognition of species with a long evolutionary history, which merit high conservation priority, will be critical for preserving geographically restricted endemics in the Himalayas and the Hengduan Mountains, where habitat loss is driving rapid declines. [ABSTRACT FROM AUTHOR]

The aim of this study was to observe the effects of remifentanil on organ damage and energy metabolism in lipopolysaccharide (LPS)-induced septic rats. A total of 45 clean-grade male Wistar rats (weight 270-320 g) were randomly divided into three groups: a control group, an LPS group, and an LPS with remifentanil treatment (LPS+REM) group. After 6 hours of modeling, the levels of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in lung and kidney tissues of rats in each group were detected by ELISA. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in lung and kidney tissues were determined, and the content of lactic acid, pyruvate and epinephrine in heart and kidney tissues were detected. Reverse transcription polymerase chain reaction and the Western blot test were used to detect the expression of pyruvate dehydrogenase kinase 4 (PDK4) in the myocardial tissue. We found that remifentanil treatment inhibited the levels of IL-6, TNF-α, and MDA in the lung and kidneys 6 h after the administration of LPS and increased the level of SOD activity. Treatment with remifentanil reduced the expression of lactic acid, pyruvate, and epinephrine in the heart and kidney tissues and attenuated the expression of PDK4 messenger RNA and PDK4 protein in the myocardial tissue. We concluded that remifentanil mignt inhibit the release of tissue inflammatory factors, regulate the body's energy metabolism, and ultimately protect the sepsis tissue damage caused by LPS. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To detect differentially expressed micro ribonucleic acids (miRNAs) in rats with myocardial ischemia/reperfusion (MIR), and to explore the influence of miR- 19a on MIR rats and its mechanism. MATERIALS AND METHODS: Firstly, the Sprague-Dawley (SD) rats were used to prepare MIR models, RNAs were extracted, and miRNA sequencing analysis was carried out to determine differentially expressed miRNAs related to MIR. Secondly, the predicted target genes of miR-19a were collected, and WebGestalt was applied to analyze gene ontology (GO) and pathway enrichment. Thirdly, the expression of the related proteins and the apoptosis of myocardial cells in MIR rats were detected via Western blotting. Fourthly, the interaction between miR- 19a and the target gene phosphatase and tensin homolog (PTEN) was examined through Luciferase reporter assay. RESULTS: Compared with that in the Sham operation (Sham) group, the miR-19a expression in rat myocardial tissues in the MIR group was significantly increased (p<0.05). Compared with those in the miR-negative control (miR-NC) group, the messenger RNA (mRNA) and protein expressions of PTEN in the miR-19a group were notably decreased (p<0.05). In comparison with the miR-NC group, miR-19a group had elevated expression of phosphorylated protein kinase B (p-Akt) (p<0.05). The Luciferase reporter gene assay manifested the direct binding of miR-19a to PTEN mRNA. CONCLUSIONS: MiR-19a inhibits the PTEN expression by directly binding to the 3'-UTR of PTEN mRNA, thus activating the Akt/p-Akt signaling pathway to suppress the apoptosis of myocardial cells in MIR injury. [ABSTRACT FROM AUTHOR]

As the water resource management progresses rapidly in recent years, middle and long-term runoff forecast has become increasingly important. Conventional multi-category runoff prediction usually utilizes manually specified threshold values to categorize runoff categories. However, this approach is arguably subjective, and it neglects fuzziness and peculiarity of hydrometeorological time series. To address this issue, a new concept, forecast domain, is proposed in this study. Cluster analysis of runoff time series was carried out with the Gaussian Mixture Model, and Support Vector Classification was then used to establish the nonlinear relationships between forecast domain and various potential predictors. The current study focuses on the Danjiangkou Reservoir, the source of the Central Route of the South-North Water Transfer Project in China. We use the 25-year data (1981-2005) for model training, and the Danjiangkou runoff data during last 11 years (2006-2016) are used for model validation. It is shown that the runoff forecast domain obtained from the unsupervised clustering is reasonable and appropriate for categorizing runoff categories. Further forecast experiments reveal that this model may shed some light on the prediction of annual mean runoff at the Danjiangkou Reservoir. [ABSTRACT FROM AUTHOR]

OBJECTIVE: This study sought to investigate the expression of interferon-λ2 (IFN-λ2) in patients with combined allergic rhinitis and nasal polyps (AR+NP), analyze the correlation between IFN-λ2 and tryptase, interleukin 10 (IL-10), and interleukin 12 (IL-12), and identify its peripheral blood cell origins. PATIENTS AND METHODS: ELISA kits were used to investigate plasma levels of IFN-λ2, tryptase, IL-10, and IL-12 in AR+NP patients and healthy controls (HC). Flow cytometry analysis was carried out to detect IFN-λ2 expression in peripheral blood leukocytes. Immunocytochemical staining was performed to detect nasal polyp IFN-λ2 expression in AR+NP patients. RESULTS: Elevated plasma IFN-λ2 levels and positive correlations between plasma IFN-λ2 and tryptase levels in AR+NP patients indicated that IFN-λ2 likely contributes to AR+NP pathogenesis. IFN-λ2 expression was upregulated in cytotoxic T cells and eosinophils in AR+NP patients. Nasal polyp mast cells and macrophages in AR+NP patients expressed IFN-λ2. CONCLUSIONS: The close correlation between IFN-λ2 expression and AR+NP may provide experimental evidence for a possible effect of IFN-λ2 against the allergic inflammatory reaction. Therefore, IFN-λ2 actions may have a potential utility for the treatment and prevention of AR+AP. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To detect the expressions of nasopharyngeal carcinoma-associated gene 6 (NGX6) in nasopharyngeal carcinoma cells and tissues, and to investigate the effects of NGX6 on the proliferation and invasion of nasopharyngeal carcinoma cells and the survival of patients. PATIENTS AND METHODS: The human nasopharyngeal carcinoma cells (HONE1) and immortalized human nasopharyngeal epithelial cells (NP69) were selected and cultured. The mRNA and protein expression levels of NGX6 were detected via quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. The expression of NGX6 in HONE1 was up-regulated using the gene transfection technique. Moreover, the effects of NGX6 on the proliferation and invasion capacities of HONE1 were observed via methyl thiazolyl tetrazolium (MTT) assay and Transwell assay. 50 biopsy tissue specimens of nasopharyngeal carcinoma and 20 non-neoplastic nasopharyngeal biopsy tissue specimens were collected, and the immunohistochemical method was used to detect the protein expression of NGX6 in tumor tissues of patients with esophageal carcinoma. Finally, the follow-up data of patients were recorded, Kaplan-Meier method was used for survival analysis, and the difference in survival rates was detected using the Log-rank test. RESULTS: The results of qRT-PCR and Western blot showed that the mRNA and protein expressions of NGX6 in HONE1 were significantly lower than those in nasopharyngeal carcinoma cells (NP69). After the overexpression of NGX6, the protein expression of NGX6 in HONE1 was significantly increased, but the proliferation and invasion capacities of HONE1 were significantly decreased. Besides, the immunohistochemical results revealed that the expression of NGX6 in tumor tissues of patients with nasopharyngeal carcinoma was significantly lower than that in normal tissues; the survival analysis showed that the level of NGX6 was positively correlated with the survival and prognosis of patients with nasopharyngeal carcinoma. CONCLUSIONS: NGX6 is lowly expressed in nasopharyngeal carcinoma, and it can inhibit the proliferation and invasion of nasopharyngeal carcinoma cells, whose expression is positively correlated with the survival and prognosis of patients with nasopharyngeal carcinoma. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Investigate the effect of three-dimensional conformal radiotherapy (3DCRT) on locally recurrent nasopharyngeal carcinoma (NPC) on the expression of succinate dehydrogenase B (SDHB). PATIENTS AND METHODS: Eighty-six patients diagnosed with locally recurrent NPC in our hospital were selected and divided into the control group (43 cases) and observation group (43 cases). Conventional two-dimensional radiotherapy was applied in the control group, and 3DCRT was adopted in the observation group. The curative effect of both groups was compared. RESULTS: The effective rate and the degree of alleviation of the observation group were higher than those of the control group, and the differences were statistically significant (p<0.05). There were no differences in the occurrence rate of complications from radiotherapy between the two groups (p>0.05). The survival rate and median survival time of the observation group were significantly higher than those of the control group (p<0.05). The positive expression rate of SDHB in the observation group after radiotherapy was significantly higher than that of the control group (p<0.05), and the median survival time of patients with positive expression of SDHB was significantly higher than patients with negative expression (p<0.05). CONCLUSIONS: 3DCRT applied for treatment of locally recurrent NPC was safe and effective. It also improved the positive expression rate of SDHB, which was associated with increased survival time. [ABSTRACT FROM AUTHOR]

OBJECTIVE: This study evaluates the value of continuous dynamic monitoring of intracranial pressure (ICP) in patients with hypertensive intracerebral hemorrhage to predict early postoperative complications. PATIENTS AND METHODS: Data from 80 patients treated in our hospital from February 2014 to February 2015 were analyzed. The patients all underwent decompressive craniectomies, and their ICP changes were monitored invasively and continuously for 1 to 7 days after surgery. The average blood loss during surgery for the group of patients was 65.3 ± 12.4 ml and the mean GCS score 8.7 ± 2.4. Cases were divided into three groups according to ICP values to compare early postoperative complications of the groups: a normal and mildly increased group (51 cases), a moderately increased group (19 cases) and a severely increased group (10 cases). RESULTS: T o v alidate t he a nalysis w e fi rst showed that comparisons among groups based on gender, age, systolic pressure, diastolic pressure, bleeding time, blood loss, operation time, craniectomy localization, and preoperative mannitol dosage yielded no statistically significant differences. In contrast, the following comparisons produced statistically significant differences: the comparison of postoperative Glasgow Coma Scale (GCS) scores showing that the lower intracranial pressure, the higher the GCS score; the postoperative rehemorrhage, cerebral edema and death ratios showing the higher the intracranial pressure, the higher the rehemorrhage ratio; the average ICP and the time to occurrence of rehemorrhage, cerebral edema or cerebral infarction, showing the relationship between the average ICP and the time to a complication. Patients with higher ICP averages suffered a complication of rehemorrhage within the first 9.6 ± 2.5 hours on average. Nevertheless, the comparison of GCS scores in those patients and the others showed no significant differences. CONCLUSIONS: Based on the findings, the dynamic monitoring of intracranial pressure can early and sensitively predict postoperative complications of patients with hypertensive cerebral hemorrhage, and guide the clinical intervention actively to improve the surgery outcome. [ABSTRACT FROM AUTHOR]

A total of 400 1-d-old Arbor Acres broiler chicks were raised at a recommended environmental temperature from d 1 to 20 (experimental day [ED] = ED1 to ED20). On ED21, the chicks were weighed and reallocated into 5 treatment groups, with 8 replicates of 10 birds each. The 5 treatment groups were as follows: the control group, in which chicks were housed at 22 ± 1°C and fed the basal diet, and the HS, HS-CUR50, HS-CUR100, and HS-CUR200 groups, in which chicks were housed at 34 ± 1°C for 8 h (0900-1700 h) and 22 ± 1°C for the rest time and fed the basal diet with 0, 50, 100, and 200 mg/ kg curcumin, respectively. From ED21 to ED42, the heat treatment lasted for 20 consecutive days. The results showed that heat-stressed broilers had greater (P < 0.05) average head surface and rectal temperature on ED21 and ED42 than the non-heat-stressed broilers. Diets supplied with 50 and 100 mg/kg curcumin increased (P < 0.05) the G:F compared to the heat-stressed groups. Mitochondrial malondialdehyde levels, an index of lipid peroxidation, in the breast muscle were 15.15 and 9.09% higher (P < 0.05) in 50 and 100 mg/kg curcumin supplemented groups than that of the heat-stressed group, respectively. Curcumin supplementation (50, 100, and 200 mg/kg) increased (P < 0.05) mitochondrial glutathione content and glutathione peroxidase, glutathione S-transferase, and manganese superoxide dismutase activities compared to heat-stressed broilers. Curcumin supplementation (50, 100, and 200 mg/kg) resulted in a decrease (P < 0.05) of heat shock protein 70 mRNA levels in the breast muscle. The breast muscle mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1α and nuclear respiratory factor 1 and 2 in heat-stressed groups was increased (P < 0.05) in response to dietary 100 mg/kg curcumin treatment. Additionally, when compared to the heat-stressed group, mitochondrial transcription factor A mRNA levels were increased (P < 0.05) by 17.64% in the 200 mg/kg curcumin supplemented group. In conclusion, dietary curcumin supplementation prevented heat-stress-impaired growth performance, possibly through improving the antioxidant defense system and enhancing the mitochondrial biogenesis. [ABSTRACT FROM AUTHOR]

Human peripheral blood monocytes are a heterogeneous population, including CD14CD16'classical' monocytes and CD14CD16'proinflammatory' monocytes. CD16 monocytes are expanded in various inflammatory conditions. However, little is known about the CD14CD16 monocytes in patients with breast cancer. We detected CD14CD16 monocytes in 96 patients with breast cancer and 54 control subjects using flow cytometry. Receiver-operating characteristic (ROC) curve analysis was used to determine the feasibility of CD14CD16 monocytes as an indicator for diagnosis of breast cancer. We found that the frequency of CD14CD16 monocytes showed a significantly greater increase in breast cancer patients than in controls (16·96% versus 10·84%, P < 0·0001). The area under the ROC curve for CD14CD16 monocytes was 0·805 [95% confidence interval (95% CI): 0·714-0·877, P = 0·0001]. Furthermore, the levels of CD16 monocytes were significantly negatively associated with the tumour size and pathological staging. In vitro, we showed that CD14CD16 monocytes were expanded significantly when the purified CD14 monocytes were exposed to Michigan Cancer Foundation (MCF)-7 cells-conditioned medium (MCF-CM) or, separately, to monocyte chemotactic protein 1 (MCP-1). Neutralizing antibodies against MCP-1 inhibited the expansion of CD14CD16 monocytes by MCF-CM. Collectively, our findings indicated that MCP-1 can expand CD14CD16 monocytes in patients with breast cancer. Furthermore, the CD14CD16 monocyte may be a useful indicator in early diagnosis of breast cancer. [ABSTRACT FROM AUTHOR]

The article discusses a study concerning the changes in the behavior and temperature of bees namely Apis mellifera and Apis cerana cerana during resting and heat-balling. According to the study, thoracic temperature of bees rose to 29.8 degree Celsius during wasp-hawking and if it persisted, 30 guard bees made a ball around the wasp and raised their temperatures to 46 degree Celsius. It further states that this temperature was above the lethal limit of the wasp and led to its death.

This study was conducted to investigate the effect of Bacillus amyloliquefaciens (BA) on the immune function of broilers challenged with lipopolysaccharide (LPS). 192 one-day-old male Arbor Acre broiler chickens were randomly distributed into four treatments: 1) broilers fed a basal diet; 2) broilers fed a basal diet supplemented with BA; 3) LPS-challenged broilers fed a basal diet; and 4) LPSchallenged broilers fed a basal diet supplemented with BA. Each treatment consisted of six replicates with eight broilers per replicate. Broilers were intraperitoneally injected with either 500 fig LPS per kg body weight or sterile saline at 16, 18 and 20 d of age. LPS decreased the average daily gain (ADG, P = 0.001) and average daily feed intake (P = 0.001). The decreased ADG (P = 0.009) and increased feed conversion ratio (P = 0.047) in LPS-challenged broilers were alleviated by BA. LPS increased the relative spleen weight (P = 0.001). Relative spleen (P = 0.014) and bursa (P = 0.024) weights in the LPS-challenged broilers were reduced by BA. LPS increased white blood cell (WBC) numbers (P = 0.001). However, the WBC numbers (P = 0.042) and the ratio of lymphocytes to WBC (P = 0.020) in LPS-challenged broilers were decreased with BA treatment. LPS decreased plasma lysozyme activity (P = 0.001), but increased concentrations of plasma corticosterone (P = 0.012) and IL-2 (P = 0.020). In contrast, BA increased lysozyme activity in plasma (P = 0.040). LPS increased mRNA abundances of splenic toll-like receptor 4 (P = 0.046), interferon γ (P = 0.008), IL-1β (P = 0.045) and IL-6, (P = 0.006). IL-2 (P = 0.014) and IL-6 (P = 0.074) mRNA abundances in LPSchallenged broilers were reduced by BA, although BA had an opposite effect for IL-10 mRNA expression in those broilers (P = 0.004). In conclusion, BA supplementation could partially alleviate the compromised growth performance and immune status of broilers under immune stress induced by LPS challenge at early age. [ABSTRACT FROM AUTHOR]

Background: Fibrin sealant is frequently used in knee surgery as an adjuvant method for reducing postoperative bleeding, however, there is no consensus regarding the efficacy of fibrin sealant., Hypothesis: Fibrin sealant achieves better efficacy in terms of blood loss control, transfusion rate and units in knee surgery compared with controls., Methods: A search of the Cochrane Collaboration (2013 Issue 09), Embase (1974-2013.09), PubMed (1966-2013.09) and Chinese databases (up to 2013.09) were conducted. The Cochrane Collaboration's tool was used to assess for bias and data were analyzed by RevMan 5.29 software., Results: This study included nine RCTs and four prospective comparative trials with a total of 1299 patients. Compared to the control, fibrin sealant achieved a decrease in hemoglobin reduction [MD=1.14, 95% CI (0.61-1.67)], transfusion rate [OR=0.36, 95% CI (0.25-0.51)], transfusion units [MD=0.47, 95% CI (0.24-0.71)], hospital stay [MD=2.22, 95% CI (0.56-3.88)] and the incidence of complications [OR=0.56, 95% CI (0.38-0.83)]. And it also reduced total blood loss, while there was no significant difference [MD=155.83, 95% CI (-525.02-213.15)]., Conclusion: Patients undergoing knee surgery would benefit from high-dose fibrin sealant with reduced transfusion rate and unit, hospital stay and complications, while they might benefit little from it in total blood loss. However, the effects of a low-dose of fibrin in knee surgery remain inconclusive., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Bulk ultrafine grained (UFG)/nanocrystal metals possess exceptional strength but normally poor ductility and thermal stability, which hinder their practical applications especially in high-temperature environments. Through microalloying strategy that enables the control of grains and precipitations in nanostructured regime, here we design and successfully produce a highly microstructure-stable UFG Al-Cu-Sc alloy with ~275% increment in ductility and simultaneously ~50% enhancement in yield strength compared with its Sc-free counterpart. Although the precipitations in UFG alloys are usually preferentially occurred at grain boundaries even at room temperature, minor Sc addition into the UFG Al-Cu alloys is found to effectively stabilize the as-processed microstructure, strongly suppress the θ-Al2Cu phase precipitation at grain boundary, and remarkably promote the θ'-Al2Cu nanoparticles dispersed in the grain interior in artificial aging. A similar microalloying strategy is expected to be equally effective for other UFG heat-treatable alloys.

The redox potentials have been determined for nine azaanthraquinones in phosphate buffer at pH 7.2 by means of cyclic voltammetry. A definite correlation has been found between the redox potentials and the inhibitory effects of the azaanthraquinones on Epstein-Barr virus early antigen (EBV-EA) activation. It has further been shown that the correlation can be made better by introducing an electronic property, i.e., the atomic charge at O11 as an additional parameter.

Our laboratory has shown that human liver microsomes metabolize the anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) via a P450-type reductive reaction to a toxic metabolite 3'-amino-3'-deoxythymidine (AMT). In the present study, we examined the role of specific human P450s and other microsomal enzymes in AZT reduction. Under anaerobic conditions in the presence of NADPH, human liver microsomes converted AZT to AMT with kinetics indicative of two enzymatic components, one with a low Km (58-74 microM) and Vmax (107-142 pmol AMT formed/min/mg protein) and the other with a high Km (4.33-5.88 mM) and Vmax (1804-2607 pmol AMT formed/min/mg). Involvement of a specific P450 enzyme in AZT reduction was not detected by using human P450 substrates and inhibitors. Antibodies to human CYP2E1, CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2A6 were also without effect on this reaction. NADH was as effective as NADPH in promoting microsomal AZT reduction, raising the possibility of cytochrome b5 (b5) involvement. Indeed, AZT reduction among six human liver samples correlated strongly with microsomal b5 content (r2 = 0.96) as well as with aggregate P450 content (r2 = 0.97). Upon reconstitution, human liver b5 plus NADH:b5 reductase and CYP2C9 plus NADPH:P450 reductase were both effective catalysts of AZT reduction, which was also supported when CYP2A6 or CYP2E1 was substituted for CYP2C9. Kinetic analysis revealed an AZT Km of 54 microM and Vmax of 301 pmol/min for b5 plus NADH:b5 reductase and an AZT Km of 103 microM and Vmax of 397 pmol/min for CYP2C9 plus NADPH:P450 reductase. Our results indicate that AZT reduction to AMT by human liver microsomes involves both b5 and P450 enzymes plus their corresponding reductases. The capacity of these proteins and b5 to reduce AZT may be a function of their heme prothestic groups.

Mitogen-activated protein (MAP) kinases are involved in many cellular processes. Here we describe the cloning and characterization of a new MAP kinase, p38-2. p38-2 belongs to the p38 subfamily of MAP kinases and shares with it the TGY phosphorylation motif. The complete p38-2 cDNA was isolated by polymerase chain reaction. It encodes a 364-amino acid protein with 73% identity to p38. Two shorter isoforms missing the phosphorylation motif were identified. Analysis of various tissues demonstrated that p38-2 is differently expressed from p38. Highest expression levels were found in heart and skeletal muscle. Like p38, p38-2 is activated by stress-inducing signals and proinflammatory cytokines. The preferred upstream kinase is MEK6. Although p38-2 and p38 phosphorylate the same substrates, the site specificity of phosphorylation can differ as shown by two-dimensional phosphopeptide analysis of Sap-1a. Additionally, kinetic studies showed that p38-2 appears to be about 180 times more active than p38 on certain substrates such as ATF2. Both kinases are inhibited by a class of pyridinyl imidazoles. p38-2 phosphorylation of ATF2 and Sap-1a but not Elk1 results in increased transcriptional activity of these factors. A sequential kinetic mechanism of p38-2 is suggested by steady state kinetic analysis. In conclusion, p38-2 may be an important component of the stress response required for the homeostasis of a cell.

The aim of the present study was to characterize human CYP2E1 turnover and examine the possible proteolytic pathways responsible for the rapid degradation of CYP2E1 in a transfected HepG2 cell line expressing human CYP2E1. Two methods were used to study the CYP2E1 turnover; after addition of cycloheximide, the half-life of the CYP2E1 in the intact cells was about 6 h as detected by PNP catalytic activity assay and immunoblot analysis of apoprotein content. CYP2E1 substrates or ligands such as 4-methylpyrazole, ethanol, glycerol, and dimethyl sulfoxide protected CYP2E1 against this rapid degradation, whereas CCl4 accelerated this process. The second procedure involved pulse-chase experiments after labeling CYP2E1 with [35S]methionine and immunoprecipitation with anti-human CYP2E1 IgG. The half-life of CYP2E1 was about 2.5 h, and the various substrates or ligands modified the turnover process within intact cells as described for the cycloheximide experiments. More than 20 different reagents including antioxidants, physiological metabolites, lysosomal inhibitors, and protease inhibitors were screened for possible effects on CYP2E1 proteolytic degradation. Dibutyryl cAMP had no effect on CYP2E1 activity or turnover. Among those reagents tested so far, the serine protease inhibitor 1-chloro-3-tosylamido-7-amino-2-heptanone hydrochloride exhibited some protection against CYP2E1 degradation. To demonstrate whether the proteasome complex is involved in this process, Czb-Ile-Glu(OtBu)-Ala-leucinal (PSI) as a cell penetrating aldehydic proteasome inhibitor and Czb-Leu-norleucinal (calpeptin inhibitor) as an aldehydic nonproteosomal protease inhibitor were used to examine their effect on both the normal and the CCl4-stimulated CYP2E1 proteolytic degradation pathways. Treatment with PSI at concentrations ranging from 5 to 80 microM resulted in a dose-dependent protection against the loss of both the normal CYP2E1 and the CCl4-modified CYP2E1. The maximum protection by PSI at a concentration of 80 microM after a 12-h chase period was about 60% in cells treated with 2 mM CCl4 or 75% in cells without CCl4 treatment. Calpeptin inhibitor afforded little or no protection against CYP2E1 degradation in the absence or presence of CCl4. PSI did not inhibit CYP2E1 catalytic activity, suggesting that it was not a ligand for CYP2E1. These results indicate that human CYP2E1 has a short half-life span and that substrates can significantly modify its turnover rate in intact HepG2 cells. The proteasome proteolytic pathway may be involved in the degradation process of both the normal and the CCl4-modified human CYP2E1 in this model.

Glycerol is widely used to stabilize cytochrome P-450 and prevent its transformation to cytochrome P-420. The effect of glycerol on the content and activity of human cytochrome P-4502E1 (CYP2E1) in a HepG2 cell line that stably and constitutively expresses this P-450 was evaluated by immunoassays and oxidation of p-nitrophenol. Addition of 100 to 200 mM glycerol to the culture medium resulted in a 2 1/2- to 3-fold increase in the content and activity of CYP2E1 in microsomes isolated from the cells. Increases could be observed within 4 to 8 hr after addition of glycerol to the culture medium. Glycerol had no effect on the content of cytochrome b5 or activities of NADPH-cytochrome P-450 reductase or NADH-cytochrome b5 reductase. Upon the addition of cycloheximide to stop protein synthesis, CYP2E1 content and activity decreased with apparent half-lives of 6 and 4 hr, respectively. Glycerol prevented or decreased this loss of CYP2E1 content and activity. Labeling CYP2E1 with [35S]methionine, followed by pulse-chase experiments with cold methionine and immunoprecipitation of CYP2E1 indicated a half-life for CYP2E1 of approximately 3 hr. Glycerol increased the half-life to approximately 11 hr. Stabilization of CYP2E1 protein by glycerol was not additive or synergistic with the increase of CYP2E1 by ethanol or 4-methylpyrazole, suggesting that all three agents elevate CYP2E1 by a similar type of mechanism in this model. These results indicate that glycerol can interact with human CYP2E1 to stabilize it against proteolytic degradation, increasing the half-life of the enzyme and thereby elevating the content and activity of CYP2E1.

The aim of the present study was to characterize human CYP2E1 turnover and examine the possible role of the proteasome proteolytic pathway in the rapid degradation of CYP2E1 in a transfected HepG2 cell line expressing human CYP2E1. Microsomes isolated from MVh2E1-9 cells catalyzed a slow degradation of the expressed CYP2E1, which was prevented by the addition of 4-methylpyrazole, a ligand which stabilizes CYP2E1. The addition of the cytosolic fraction of the HepG2 cells to the microsomes produced rapid degradation of CYP2E1. This rapid degradation required MgATP and was completely prevented by 4-methylpyrazole. Pulse-chase experiments after labeling CYP2E1 with [35S]-methionine and immunoprecipitation with anti-human CYP2E1 IgG indicated a biphasic turnover of CYP2E1 with half-lives of 2.5 and 6 hours. The addition of Czb-Ile-Glu(OtBu)-Ala-Leucinal(PSI) as a cell penetrating proteasome inhibitor, at concentrations ranging from 5 to 80 microM resulted in protection against the degradation of CYP2E1. PSI also increased the steady state accumulation of CYP2E1, consistent with its inhibition of CYP2E1 turnover. These results suggest that the proteasome complex plays a major role in the degradation of human CYP2E1 in the transfected HepG2 cells.

NADH is reactive in interacting with iron and liver microsomes to catalyze the formation of reactive oxygen species. NADH-dependent microsomal electron transfer involves the enzymes NADH-cytochrome b5 reductase and cytochrome b5. Experiments were carried out to evaluate the ability of reconstituted systems containing purified reductase in the absence or presence of b5 to reduce several ferric complexes, to generate H2O2, and to catalyze lipid peroxidation. The reductase directly reduced ferric-EDTA; addition of b5 inhibited this reduction probably due to competition for the reductase. Cytochrome b5 was required for reduction of low (5 microM) and high (50 microM) concentrations of ferric-histidine and ferric-ammonium sulfate and low concentrations of ferric-ATP. The reductase could interact directly with high (50 microM) concentrations of ferric-ATP. Peroxidation of phospholipids extracted from liver microsomes by the reductase required b5. Molar ratios of b5 to reductase approximating those found in liver microsomes (e.g., 10) were effective in catalyzing lipid peroxidation and ferric reduction. The role of b5 in catalyzing lipid peroxidation appears to involve reduction of the ferric catalyst to help form an initiation complex and degradation of lipid hydroperoxides by the hemeprotein to catalyze propagation of the peroxidation cycle. In contrast to results with microsomes, lipid peroxidation by the complete reconstituted system was sensitive to super-oxide dismutase; this sensitivity was decreased if the reconstituted system was dialyzed overnight to form vesicular preparations, indicating that accessibility of enzymes to sites of peroxidation was important. High rates of H2O2 formation were observed in the presence of ferric-EDTA plus reductase; rates of H2O2 formation with the other ferric complexes were low even in the presence of b5. These results indicate that the ability of NADH reductase and cytochrome b5 to interact with various ferric complexes depends on the nature of the chelating agent used to complex the iron and on the concentration of the iron.

Mitogen-activated protein kinases are members of a conserved cascade of kinases involved in many signal transduction pathways. They stimulate phosphorylation of transcription factors in response to extracellular signals such as growth factors, cytokines, ultraviolet light, and stress-inducing agents. A novel mitogen-activated protein kinase kinase, MEK6, was cloned and characterized. The complete MEK6 cDNA was isolated by polymerase chain reaction. It encodes a 334-amino acid protein with 82% identity to MKK3. MEK6 is highly expressed in skeletal muscle like many other members of this family, but in contrast to MKK3 its expression in leukocytes is very low. MEK6 is a member of the p38 kinase cascade and efficiently phosphorylates p38 but not c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) family members in direct kinase assays. Coupled kinase assays demonstrated that MEK6 induces phosphorylation of ATF2 by p38 but does not phosphorylate ATF2 directly. MEK6 is strongly activated by UV, anisomycin, and osmotic shock but not by phorbol esters, nerve growth factor, and epidermal growth factor. This separates MEK6 from the ERK subgroup of protein kinases. MEK6 is only a poor substrate for MEKK, a mitogen-activated protein kinase kinase kinase that efficiently phosphorylates the related family member JNKK.

Ethanol can be oxidized to the 1-hydroxyethyl radical (HER) by rat and deer mice liver microsomal systems. Experiments were carried out to evaluate the ability of human liver microsomes to catalyze this reaction, compare the effectiveness of NADH with that of NADPH, and assess the possible role of cytochrome b5 in HER formation. HER was detected as the alpha-(4-pyridly-1 -oxide)-N-t-butylnitrone/HER adduct. Human liver microsomes catalyzed HER formation with either NADPH or NADH as cofactor; rates with NADH were approximately 50% those found with NADPH. Chelex-100 treatment of the reaction mixture produced marked inhibition of HER formation, suggesting that a transition metal, such as iron, was required to catalyze the reaction. The addition of ferric chloride restore HER formation. Catalase (2600 units/ml) and superoxide dismutases (500 units/ml) nearly completely inhibited the reaction with either NADPH or NADH. The NADH-dependent rates of superoxide production, detected as 5,5-dimethyl-1-pyrroline-N-oxide-O2H, were approximately 50% the NADPH-dependent rates, which is consistent with the rates of HER formation. Anti-cytochrome b5 IgG decreased NADPH- and NADH-dependent HER formation, and this was associated with inhibition of superoxide formation with both reductants. These results indicate that human liver microsomes can catalyze the oxidation of ethanol of HER with either NADPH or NADH as reductant. The effectiveness of NADH may be significant in view of the increased NADH/NAD+ redox ratio in the liver as a consequence of ethanol oxidation by alcohol dehydrogenase. HER formation by human liver microsomes seems to be catalyzed by an oxidant derived from the interaction of iron with superoxide or H2O2, and a close association exists between HER formation and superoxide production. Cytochrome b5 seems to play a role in HER formation, most likely due to its effect on superoxide production.

The NADH-dependent microsomal electron transfer system consists of NADH-cytochrome b5 reductase and cytochrome b5, which donates reducing equivalents to fatty acyl desaturase, cytochrome P450, and other reactions. A study was carried out to investigate the interaction of NADH with several ferric complexes and to evaluate the role of cytochrome b5 in these interactions. NADH-dependent microsomal lipid peroxidation was stimulated by ferric-ATP, ferric-histidine, and ferric-ammonium sulfate, but not by ferric-EDTA. Anti-cytochrome b5 IgG produced a concentration-dependent inhibition of lipid peroxidation catalyzed by all three ferric complexes. Addition of purified cytochrome b5 to the microsomes increased the rate of lipid peroxidation with all three ferric complexes. Lipid peroxidation in control and the cytochrome b5-fortified microsomes was not sensitive to superoxide dismutase, catalase, or DMSO and was completely inhibited by trolox and propylgallate. Ferric-EDTA stimulated NADH-dependent microsomal production of H2O2 and NADH consumption. Anti-cytochrome b5 IgG had only a small inhibitory effect on this stimulation by ferric-EDTA. NADH supported microsomal reduction of ferric complexes in the order ferric-ATP > ferric-histidine approximately ferric-ammonium sulfate > ferric-EDTA. Anti-cytochrome b5 IgG inhibited, whereas added cytochrome b5 stimulated, the reduction of ferric-ATP, ferric-histidine, and ferric-ammonium sulfate, whereas reduction of ferric-EDTA was not affected by these additions. Ferric-ATP, at high concentrations, was more effective than ferric-histidine or ferric-ammonium sulfate in stimulating lipid peroxidation and in becoming reduced by NADH-dependent microsomal electron transport; anti-cytochrome b5 IgG was less inhibitory and added b5 was less stimulatory at 50 microM ferric-ATP compared to 5 microM ferric-ATP or 50 microM ferric-histidine or 50 microM ferric-ammonium sulfate. It is concluded that cytochrome b5 is required for reduction of low and high concentrations of ferric-histidine and ferric-ammonium sulfate and low concentrations of ferric-ATP and for the lipid peroxidation catalyzed by these ferric complexes. The reductase, not cytochrome b5, is involved in interaction with ferric-EDTA. Higher concentrations of ferric-ATP can also interact with the reductase, as well as with cytochrome b5.

Bone metabolism is regulated by a balance between bone resorption caused by osteoclasts and bone formation caused by osteoblasts. This balance is disturbed in postmenopausal women as a result of lower serum estrogen levels. Estrogen, which is used in hormone replacement therapy to prevent postmenopausal osteoporosis, downregulates expression of the interleukin 6 (IL-6) gene in osteoblasts and bone marrow stromal cells. IL-6 is directly involved in bone resorption by activating immature osteoclasts. We show here that NF-kappa B and C/EBP beta are important regulators of IL-6 gene expression in human osteoblasts. Importantly, the IL-6 promoter is inhibited by estrogen in the absence of a functional estrogen receptor (ER) binding site. This inhibition is mediated by the transcription factors NF-kappa B and C/EBP beta. Evidence is presented for a direct interaction between these two factors and ER. We characterized the protein sequence requirements for this association in vitro and in vivo. The physical and functional interaction depends in part on the DNA binding domain and region D of ER and on the Rel homology domain of NF-kappa B and the bZIP region of C/EBP beta. The cross-coupling between ER, NF-kappa B, and C/EBP beta also results in reduced activity of promoters with ER binding sites. We further show that the mechanism of IL-6 gene repression by estrogen is clearly different from that of activation of promoters with ER binding sites. Therefore, drugs that separate the transactivation and transrepression functions of ER will be very helpful for treatment of osteoporosis without causing undesirable side effects.

A simplified, rapid procedure for the purification of NADH-cytochrome b5 oxidoreductase and cytochrome b5 from either rat or rabbit liver is described. Microsomes were prepared by fractionation with polyethylene glycol and solubilized with Triton X-100. Cytochrome b5 was purified by a two-column procedure, anion exchange chromatography using DEAE-cellulose, and hydrophobic chromatography on phenyl-Sepharose. The final preparation of cytochrome b5 was purified more than a 120-fold from rat or rabbit liver microsomes, with specific content of about 50 nmol per mg protein, and overall yield of 22 to 32%. Only a single band with mol wt of 18,600 was found on sodium dodecyl sulfate (SDS)-gels or on Western blots using a polyclonal antibody raised against the purified b5. NADH-cytochrome b5 oxidoreductase was purified by a three-column procedure, DEAE-cellulose, hydroxylapatite, and ADP-agarose. The final product was purified more than 400-fold from rat or rabbit liver microsomes with a yield of about 25% and final specific activity of about 1600 mumol ferricyanide reduced per minute per milligram of protein. A single band with mol wt of 33, 100 was found on SDS-gels. The reductase catalyzed reduction of ferricyanide, dichlorophenol-indophenol, and cytochrome b5. Cytochrome c was reduced in the presence of reductase plus cytochrome b5, and this was inhibited by the anti-b5 IgG. The reductase catalyzed a rapid rate of reduction of ferric-ATP, which was slightly elevated by cytochrome b5. Ferric-histidine and ferric-ammonium sulfate were slowly reduced by reductase; addition of cytochrome b5 markedly stimulated reduction of these ferric complexes but inhibited reduction of ferric-EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)