Combined TGE-SGE expression of novel PAI-1-resistant t-PA in CHO DG44 cells using orbitally shaking disposable bioreactors

An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncatedmutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncatedmutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.     

Effects of hypercapnia and NO synthase inhibition in sustained hypoxic pulmonary vasoconstriction

Background

Acute respiratory disorders may lead to sustained alveolar hypoxia with hypercapnia resulting in impaired pulmonary gas exchange. Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange during local acute (0-30 min), as well as sustained (> 30 min) hypoxia by matching blood perfusion to alveolar ventilation. Hypercapnia with acidosis improves pulmonary gas exchange in repetitive conditions of acute hypoxia by potentiating HPV and preventing pulmonary endothelial dysfunction. This study investigated, if the beneficial effects of hypercapnia with acidosis are preserved during sustained hypoxia as it occurs, e.g in permissive hypercapnic ventilation in intensive care units. Furthermore, the effects of NO synthase inhibitors under such conditions were examined.

Method

We employed isolated perfused and ventilated rabbit lungs to determine the influence of hypercapnia with or without acidosis (pH corrected with sodium bicarbonate), and inhibitors of endothelial as well as inducible NO synthase on acute or sustained HPV (180 min) and endothelial permeability.

Results

In hypercapnic acidosis, HPV was intensified in sustained hypoxia, in contrast to hypercapnia without acidosis when HPV was amplified during both phases. L-N^G-Nitroarginine (L-NNA), a non-selective NO synthase inhibitor, enhanced acute as well as sustained HPV under all conditions, however, the amplification of sustained HPV induced by hypercapnia with or without acidosis compared to normocapnia disappeared. In contrast 1400 W, a selective inhibitor of inducible NO synthase (iNOS), decreased HPV in normocapnia and hypercapnia without acidosis at late time points of sustained HPV and selectively reversed the amplification of sustained HPV during hypercapnia without acidosis. Hypoxic hypercapnia without acidosis increased capillary filtration coefficient (Kfc). This increase disappeared after administration of 1400 W.

Conclusion

Hypercapnia with and without acidosis increased HPV during conditions of sustained hypoxia. The increase of sustained HPV and endothelial permeability in hypoxic hypercapnia without acidosis was iNOS dependent.     

Expression Levels of the BDNF Gene and Histone Modifications Around Its Promoters in the Ventral Tegmental Area and Locus Ceruleus of Rats During Forced Abstinence from Morphine

Brain-derived neurotrophic factor (BDNF) plays a role in mediating molecular, cellular, and behavioral adaptations underlying drug addiction. Here, we examined the influence of withdrawal from repeated morphine treatment on the expression of BDNF mRNA in the ventral tegmental area (VTA) and locus coeruleus (LC) of the rat brain. We also studied whether alternations in mRNA levels of BDNF in these tissues are associated with histone modifications around promoters II and III of the BDNF gene. Thus, chromatin immunoprecipitation (CHIP) and quantitative (q)-PCR were employed to assess acetylation of histone H3 at K9/K14 and trimethylation of histone H3 at K9. Results of qRT-PCR showed that levels of BDNF mRNA in both VTA and LC were significantly increased 7 days rather than 2 h or 24 h following the last injection of morphine. Consistently, CHIP and qPCR analysis revealed that on day 7 of morphine abstinence, both VTA and LC levels of histone methylation at BDNF promoters II and III of morphine treated rats were significantly lower than control animals. Morphine withdrawal caused only a significant increase in H3 acetylation at the promoter II in the LC. These data demonstrate the involvement of histone H3 methylation in the regulation of gene expression in the VTA and LC of rats during forced abstinence of morphine.     

Large-scale virtual screening for the identification of new Helicobacter pylori urease inhibitor scaffolds

Here, we report a structure-based virtual screening of the ZINC database (containing about five million compounds) by computational docking and the analysis of docking energy calculations followed by in vitro screening against H. pylori urease enzyme. One of the compounds selected showed urease inhibition in the low micromolar range. Barbituric acid and compounds 1a, 1d, 1e, 1f, 1g, 1h were found to be more potent urease inhibitors than the standard inhibitor hydroxyurea, yielding IC(50) values of 41.6, 83.3, 66.6, 50, 58.8, and 60 μM, respectively (IC(50) of hydroxyurea = 100 μM). 5-Benzylidene barbituric acid has enhanced biological activities compared to barbituric acid. Furthermore, the results indicated that among the substituted 5-benzylidene barbiturates, those with para substitution have higher urease inhibitor activities. This may be because the barbituric acid moiety is closer to the bimetallic nickel center in unsubstituted or para-substituted than in ortho- or meta-substituted analogs, so it has greater chelating ability.     

Association of cyclin-dependent kinase inhibitor 2A/B with increased risk of developing breast cancer

There is a growing body of data reporting the association of genetic alterations in chromosome 9P21 with the risk of developing cancer. In the current study, we studied the association of a genetic variant in CDKN2A/B, rs1333049, with the risk of developing breast cancer. A total of 339 participants with and without breast cancer entered to the study. Genotyping was done by the TaqMan real-time polymerase chain reaction (RT-PCR) method and gene expression analysis was ran by RT-PCR. Our data showed that the minor allele homozygote in the total population was 10%, whereas for heterozygote was 38%. The dominant genetic model demonstrated that individuals with breast cancer had advanced TNM classification. Moreover, the logistic regression revealed that individuals who had CC/CG genotypes might have an enhanced risk of developing breast cancer when compared to the holders of GG genotype (e.g., OR = 2.8; 95% CI,1.4–5.4; p = .001), after regulated for confounders; age and body mass index. Furthermore, our analysis showed that the CDKN2A/B gene was downregulated in patients (p < .001). We showed a meaningful relationship of CDKN2A/B with the risk of breast cancer, cancer, showing the importance of studies in great sample size and several centers for studying the value of the marker as a risk classification in the management of patients with breast cancer.     

Protein kinase C mediates the hormonally regulated plasma membrane fusion of avian embryonic skeletal muscle

Recent findings have demonstrated that prostanoid-generated calcium fluxes can trigger myoblast fusion and suggest inositol phospholipid turnover as part of the fusion mechanism. Here we demonstrate that a block imposed on myoblast fusion by antagonists of prostanoid biosynthesis can be overcome by either the membrane-permeable diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG), or 12-O-tetradecanoylphorbol 13-acetate (TPA). Both phorbol and the membrane-impermeable dioleoylglycerol were ineffective. These results implicate protein kinase C activation in prostaglandin E1-mediated myoblast fusion and add weight to the contention that inositol turnover is involved in the regulation of myoblast fusion.     

DMSO and retinoic acid induce HL-60 differentiation by different but converging pathways

The expression of c-myc and two calcium-binding proteins, MRP8 and MRP14, has been analyzed in wild-type and differentiation-resistant HL-60 variants. In HL-R5 cells, resistant to the induction of differentiation by retinoic acid but not DMSO, the characteristic c-myc down-regulation which is associated with HL-60 differentiation, as well as increased levels of MRP8 and MRP14, is detectable only after DMSO treatment. By contrast HL-D4 cells, which were selected for resistance to the induction of differentiation by DMSO alone, are actually resistant to both DMSO and retinoic acid. However, treatment of HL-D4 cells with DMSO results in a transient c-myc down-regulation in the absence of either growth arrest or induction of differentiation. Neither agent can induce an increase in the level of either MRP8 or MRP14 in HL-D4. The resistance of HL-D4 cells to DMSO and retinoic acid, and the different effects of these agents on c-myc RNA levels, despite their common effect on the expression of MRP8 and MRP14, suggest that the two agents act through different pathways which coverage before the onset of myeloid differentiation in HL-60 cells.     

Low Temperature and Long Residence Time AFEX Pretreatment of Corn Stover

Low temperature and long residence time pretreatments have been proposed as an alternative to conventional pretreatments within a centralized biorefinery, allowing for a decentralized pretreatment without high energy costs. Ammonia fiber expansion (AFEX?) pretreatment may be uniquely suitable for decentralized pretreatment, and this study considers the possibility of decreasing the temperature in AFEX pretreatment of corn stover. AFEX pretreatment at 40°C and 8?h produced comparable sugar and ethanol yields as conventional AFEX pretreatment at high temperatures and short residence time during subsequent hydrolysis and fermentation. Increasing the ammonia loading at these temperatures tends to increase digestibility, although the moisture content of the reaction has little effect. This study suggests a greater flexibility in AFEX pretreatment conditions than previously thought, allowing for an alternative approach for decentralized facilities if the economic conditions are appropriate.