Regulatory mechanism of Haa1p and Tye7p in Saccharomyces cerevisiae when fermenting mixed glucose and xylose with or without inhibitors.

BACKGROUND: Various inhibitors coexist in the hydrolysate derived from lignocellulosic biomass. They inhibit the performance of Saccharomyces cerevisiae and further restrict the development of industrial bioethanol production. Transcription factors are regarded as targets for constructing robust S. cerevisiae by genetic engineering. The tolerance-related transcription factors have been successively reported, while their regulatory mechanisms are not clear. In this study, we revealed the regulation mechanisms of Haa1p and Tye7p that had outstanding contributions to the improvement of the fermentation performance and multiple inhibitor tolerance of S. cerevisiae. RESULTS: Comparative transcriptomic analyses were applied to reveal the regulatory mechanisms of Haa1p and Tye7p under mixed sugar fermentation conditions with mixed inhibitors [acetic acid and furfural (AFur)] or without inhibitor (C) using the original strain s6 (S), the HAA1-overexpressing strain s6H3 (H), and the TYE7-overexpressing strain s6T3 (T). The expression of the pathways related to carbohydrate, amino acid, transcription, translation, cofactors, and vitamins metabolism was enhanced in the strains s6H3 and s6T3. Compared to C_H vs. C_S group, the unique DEGs in AFur_H vs. AFur_S group were further involved in oxidative phosphorylation, purine metabolism, vitamin B6 metabolism, and spliceosome under the regulation of Haa1p. A similar pattern appeared under the regulation of Tye7p, and the unique DEGs in AFur_T vs. AFur_S group were also involved in riboflavin metabolism and spliceosome. The most significant difference between the regulations of Haa1p and Tye7p was the intracellular energy supply. Haa1p preferred to enhance oxidative phosphorylation, while Tye7p tended to upregulate glycolysis/gluconeogenesis. CONCLUSIONS: Global gene expressions could be rewired with the overexpression of HAA1 or TYE7. The positive perturbations of energy and amino acid metabolism were beneficial to the improvement of the fermentation performance of the strain. Furthermore, strengthening of key cofactor metabolism, and transcriptional and translational regulation were helpful in improving the strain tolerance. This work provides a novel and comprehensive understanding of the regulation mechanisms of Haa1p and Tye7p in S. cerevisiae.

[Regulatory function of Shen-reinforcing and qi-supplementing drugs on hypothalamic-pituitary-adrenal axis and interleukin-6 dysfunction in asthmatic rats].

OBJECTIVE: To evaluate the effect of Shen-reinforcing and qi-supplementing (SRQS) drugs on some ingredients of neuro-endocrine-immune (NEI) network in asthma rat model. METHODS: Asthma model was established by ovalbumin sensitization and long-term excitation. Forty healthy Brown Norway rats of clean grade were randomly divided into 4 groups by randomized digital table, the normal control group and the three treated groups treated by low, moderate and high dose of SRQS drugs respectively. Blood content of adrenocorticotrophic hormone (ACTH) was detected by RIA; interleukin-6 (IL-6) and corticosterone were determined by ELISA; and the mRNA expresion of corticosteroid release hormone (CRH) in hypothalamus was tested by Realtime-PCR. RESULTS: Eosinophile inflammation was shown in the pathology of asthma model rats, and also shown a multiple level hypothalamic-pituitary-adrenal axis (HPA axis) disorder at the repeated attack of asthma. After treatment, levels of ACTH and CRH mRNA expression in the treated groups were significantly higher than those in the control group (P <0.05), but the corticosterone only showed a rising tendency. Level of IL-6 increased during the episode, showing a significant negative correlation with ACTH (r = - 0.325, P = 0.043), and had somewhat reduction after SRQS treatment. CONCLUSION: SRQS drugs could improve the function of HPH axis independent of IL-6, suggesting that the action is possibly targeted on the neuro-endocrine axis, which might be the hypothalamus.

[Effect of epimedium herb on RANTES and monocyte chemotactic protein-3 expression in lung tissue of asthmatic rats].

OBJECTIVE: To observe the effects of epimedium herb (EH), a Chinese herb for replenishing Shen, on regulated on activation, normal T call expressed and secreted (RANTES) and monocyte chemotactic protein-3 (MCP-3) expression in lung tissue of asthmatic rats, for further exploring the action mechanism of EH in treating asthma. METHODS: Brown Norway rats were randomly divided into six groups: the normal control group (A), the allergic asthma model group (B), the group of model rat treated with dexamethasone (C), and the three groups of model rat treated with low (0.125 g/mL), medium (0.5 g/mL), high (2.0 g/mL) dose of EH (D, E and F). RANTES and MCP-3 mRNA expressions in lung tissue were tested with Real-time PCR and the serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin-4 (IL-4), and interleukin-5 (IL-5) were measured by enzyme linked immunosorbent assay (ELISA). RESULTS: As compared with the model group (B), RANTES, MCP-3 expression and TNF-alpha were lower in the 4 treated groups (C, D, E and F, P < 0.05 or P < 0.01); IL-4 was lower in Group C, E and F (P = 0.007, P = 0.047, P = 0.033), while that in Group D was higher than that in Group C (P = 0.012). As for level of IL-5, lowering was shown only in Group C and F (P = 0.003, P = 0.005). CONCLUSION: Applying EH in the attack stage of asthma can alleviate the airway inflammation by down-regulating the expression of RANTES and MCP-3 in lung tissue.

[The effects of Ad-Gax transfection on apoptosis and related gene expression in pulmonary arterial smooth muscle cells in hypoxic rat model].

OBJECTIVE: To explore the effects of growth arrest-specific homeobox (Gax) transfection on apoptosis and expressions of Bcl-2 and Bax proteins of rat pulmonary arterial smooth muscle cells (PASMCs) exposed to hypoxia. METHODS: PASMCs were transfected with Gax gene by a replication-deficient adenovirus expressing the hemagglutinin-tagged Gax cDNA (Ad-Gax). After exposure to normal oxygenation (21% O(2)) or hypoxia (2.5% O(2)) for 2 h, 6 h, 12 h, 24 h and 48 h, apoptosis was observed by transmission electronic microscope and TUNEL-positive staining. The expressions of Bcl-2 and Bax proteins in PASMCs were detected by immunocytochemistry. RESULTS: Before Ad-Gax transfection, none or few of TUNEL-positive PASMCs were detected. After Ad-Gax transfection, the PASMCs unexposed to hypoxia displayed none or few TUNEL-positive stain, while the PASMCs exposed to hypoxia displayed a marked increase in TUNEL-positive stain, especially in cells exposed to hypoxia for 24 h to 48 h. The ratio of apoptosis of PASMCs at normoxic, hypoxia 2 h, 6 h, 12 h, 24 h and 48 h were (9.11 +/- 1.21)%, (34.13 +/- 1.02)%, (39.12 +/- 0.43)%, (50.09 +/- 0.13)%, (60.04 +/- 1.12)% and (55.47 +/- 0.03)% (P < 0.01), respectively. Before Ad-Gax transfection, the level of Bax protein showed no significant increase in PASMCs exposed to hypoxia, while that of Bcl-2 protein increased significantly, as compared to that in PASMCs under normoxic condition. The average optical density (AOD) of Bcl-2 was 2.31 +/- 0.12, 2.35 +/- 0.23, 2.49 +/- 0.27, 2.51 +/- 0.19, 2.54 +/- 0.25 and 2.53 +/- 0.20 at normoxic, hypoxia for 2 h, 6 h, 12 h, 24 h and 48 h before Ad-Gax transfection, respectively (P < 0.05, P < 0.01). After Ad-Gax transfection to PASMCs exposed to hypoxia, Bax protein increased; the AOD of Bax was 3.82 +/- 0.38, 3.12 +/- 0.42, 3.53 +/- 0.61, 4.52 +/- 0.23, 4.25 +/- 0.76 and 4.03 +/- 0.38 at normoxic, hypoxia for 2 h, 6 h, 12 h, 24 h and 48 h (P < 0.01), respectively. But Bcl-2 protein decreased significantly; the AOD of Bcl-2 was 9.11 +/- 1.21, 34.13 +/- 1.02, 39.12 +/- 0.43, 50.09 +/- 0.13, 60.04 +/- 1.12 and 55.47 +/- 0.03, respectively (P < 0.01). After Ad-Gax transfection, the Bcl-2/Bax ratio was negatively correlated with the rate of apoptotic PASMCs (r = -0.53, P < 0.01). CONCLUSION: Ad-Gax transfection induced PASMC apoptosis after exposure to hypoxia. A possible mechanism is that Gax can downregulate Bcl-2 expression and upregulate Bax expression, especially by decrease of the Bcl-2/Bax ratio.

[Effects of Chinese herbs for replenishing shen and strengthening qi on some indexes of neuro-endocrino-immune network in asthmatic rats].

OBJECTIVE: To study changes of several related indexes of neuro-endocrino-immune (NEI) network in rat asthma model, and to observe effects of epimedium herb (EH, a Chinese herb for replenishing Shen) and milkvetch root (MR, a Chinese herb for strengthening qi) on these indexes. METHODS: One hundred and twenty male healthy Brown Norway rats of clean grade were randomly divided into 11 groups. Their mRNA expression of corticoid release hormone (CRH) in hypothalamus was tested by Realtime-PCR, serum ACTH and CORT, IL-6, IL-4, IFN-gamma were detected with radioimmunoassay and enzyme-linked immunosorbent assay respectively and pathological changes of lung tissue were observed with hematoxylin and eosin stain. RESULTS: As compared with the normal group, mRNA expression of CRH in hypothalamic tissue, plasma ACTH and serum concentration of IFN-gamma not changed significantly, but serum level of CORT raised significantly and the pre-inflammatory factors IL-6 and IL-4 showed an increasing trend in the model rats. As compared with the model group, level of CRH mRNA expression in groups treated with low dose and moderate dose of both EH and the formula (HE and MR), IFN-gamma in group treated with moderate dose of EH and serum CORT in group treated with low dose of MR were higher, and serum levels of IL-4 and IL-6 in groups treated with high dose of MR were lower. CONCLUSION: Rats suffered from repeated asthmatic attack for three weeks had some disorders in indexes of NEI network. Chinese herbs for replenish Shen and strengthening qi could improve the function of hypothalamic-pituitary-adrenal axis and adjust the balance of Th1 and Th2 cytokines, which might be one of the mechanisms of Chinese herbs for treatment of repeated asthma attack.

[Effects of milkvetch root on neuro-endocrino-immune network in asthma model rat].

OBJECTIVE: To study the change and the effect of milkvetch root (MR) on neuro-endocrino-immune (NEI) network related indexes in repeated asthmatic attack model rats. METHODS: Rats were randomly divided into five groups: the normal group (A), the model group (B), and the three treated groups (C, D, E) treated with low, medium and high dose of the MR by gastrogavage. The corticotropin releasing hormone (CRH) mRNA expression in hypothalamus was tested by Realtime-PCR, serum adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were detected with radioimmunoassay, serum IL-6, IL-4, IFN-gamma determined with enzyme-linked immunosorbent assay, and the lung tissue pathology was examined by hematoxylin and eosin staining. RESULTS: As compared with the normal group, in the rats 3 weeks after modeling, CRH mRNA expression, blood IFN-gamma and plasma ACTH were unchanged, serum level of CORT raised significantly (P<0.05), IL-6 and IL-4 showed an increasing trend but without significance. Low dose of MR could promote the production of serum CORT, and hight dose of MR could down-regulate the concentrations of IL-4 and IL-6 (P<0.01). No significant difference was found in comparison of pathological changes of lung tissue among the groups. CONCLUSION: Rats suffered from repeated asthmatic attack have some disorders in indexes of NEI, MR could enhance the function of hypothalamic-pituitary-adrenal cortex axis and adjust the balance of Th1 and Th2 cytokines to alleviate the inflammation of asthma.

[Effects on herba epimedii and radix Astragali on tumor necrosis factor-alpha and nuclear factor-kappa B in asthmatic rats].

OBJECTIVE: To observe the effects of Herba Epimedii and Radix Astragali, the two Chinese herbs for replenishing Shen and strengthening qi, on tumor necrosis factor-alpha (TNF-alpha, one of the pro-inflammatory factors) and nuclear factor-kappa B (NF-kappa B) in asthmatic rats. METHODS: Rats were randomly divided into five groups: the normal saline control group, the asthma model group and the three treated groups treated with high, medium and low dose of the Chinese herbs. Serum TNF-alpha and NF-kappa B activity in pulmonary tissue were detected with enzyme-linked immunosorbent assay and immunohistochemistry respectively. RESULTS: Herba Epimedii and Radix Astragali could effectively reduce the production of TNF-alpha and inhibit NF-kappa B activity, and the efficacies in the three treated group were similar, showing insignificant difference among them. CONCLUSION: Application of Herba Epimedii and Radix Astragali in the attack or remission stage of asthma could restrain the development of inflammation by reducing the production of TNF-alpha and inhibiting NF-kappa B activity.