A potential role of protein extractions from Metagonimus yokogawai in amelionating inflammation in patients with ankylosing spondylitis.

Helminth infections and their components has been recognized to have a positive impact on the immune system. This study aimed to investigate the potential of Metagonimus yokogawai-derived proteins (MYp) to provide protection against ankylosing spondylitis (AS) through modulation of immune responses. The cytotoxicity of MYp at various doses was first assessed using MTS and flow cytometry. Peripheral blood mononuclear cells (PBMCs) were collected from AS patients, and the production of inflammatory cytokines was analyzed through flow cytometry. In the experiments with SKG mice, MYp or vehicle was administered and inflammation was evaluated through immunohistochemistry and enzyme-linked immunosorbent assay. The results showed that MYp did not decrease cell viability of PBMCs even after 48 h. Additionally, the frequencies of IFN-γ and IL-17A producing cells were significantly reduced after MYp treatment in the PBMC cultures. Furthermore, MYp treatment significantly suppressed arthritis and enthesitis in the SKG mouse model. The results suggest the first evidence that MYp can effectively alleviate clinical symptoms and restore cytokine balance in patients with AS.

CCL20 inhibition for treating inflammation in ankylosing spondylitis.

OBJECTIVES: Th17 cells are known to play a significant role in AS. C-C motif chemokine ligand 20 (CCL20) binds to C-C chemokine receptor 6 (CCR6) on Th17 cells, promoting their migration to inflammation sites. The aim of this research is to examine the effectiveness of CCL20 inhibition in treating inflammation in AS. METHODS: Mononuclear cells from peripheral blood (PBMC) and SF (SFMC) were collected from healthy individuals and AS. Flow cytometry was used to analyse cells producing inflammatory cytokines. CCL20 levels were determined using ELISA. The impact of CCL20 on Th17 cell migration was verified using a Trans-well migration assay. The in vivo efficacy of CCL20 inhibition was evaluated using an SKG mouse model. RESULTS: The presence of Th17 cells and CCL20 expressing cells was higher in SFMCs from AS patients compared with their PBMCs. The CCL20 level in AS SF was significantly higher than in OA patients. The percentage of Th17 cells in PBMCs from AS patients increased when exposed to CCL20, whereas the percentage of Th17 cells in SFMCs from AS patients decreased when treated with CCL20 inhibitor. The migration of Th17 cells was found to be influenced by CCL20, and this effect was counteracted by the CCL20 inhibitor. In the SKG mouse model, the use of CCL20 inhibitor significantly reduced joint inflammation. CONCLUSION: This research validates the critical role of CCL20 in AS and suggests that targeting CCL20 inhibition could serve as a novel therapeutic approach for AS treatment.

Bone Marrow-Derived Vasculogenic Mesenchymal Stem Cells Enhance In Vitro Angiogenic Sprouting of Human Umbilical Vein Endothelial Cells.

Vasculogenic properties of bone marrow-derived mesenchymal stem cells (MSCs) have been reported, but it is still unclear whether the vasculogenic properties are restricted to some populations of MSCs or whether the entire population of MSCs has these properties. We cultured two different populations of MSCs in different culture media and their vasculogenic properties were evaluated using In vitro spheroid sprouting assay. Neither population of MSCs expressed markers of endothelial progenitor cells (EPCs), but they were different in the profiling of angiogenic factor expression as well as vasculogenic properties. One population of MSCs expressed basic fibroblast growth factor (bFGF) and another expressed hepatocyte growth factor (HGF). MSCs expressing HGF exhibited In vitro angiogenic sprouting capacity in response to bFGF derived from other MSCs as well as to their autocrine HGF. The vasculogenic mesenchymal stem cells (vMSCs) derived from the bone marrow also enhanced In vitro angiogenic sprouting capacity of human umbilical vein endothelial cells (HUVECs) in an HGF-dependent manner. These results suggest that MSCs exhibit different vasculogenic properties, and vMSCs that are different from EPCs may contribute to neovascularization and could be a promising cellular therapy for cardiovascular diseases.

Regioselective C-H hydroxylation of omeprazole sulfide by Bacillus megaterium CYP102A1 to produce a human metabolite.

OBJECTIVES: To find a simple enzymatic strategy for the efficient synthesis of the expensive 5'-hydroxyomeprazole sulfide, a recently identified minor human metabolite, from omeprazole sulfide, which is an inexpensive substrate. RESULTS: The practical synthetic strategy for the 5'-OH omeprazole sulfide was accomplished with a set of highly active CYP102A1 mutants, which were obtained by blue colony screening from CYP102A1 libraries with a high conversion yield. The mutant and even the wild-type enzyme of CYP102A1 catalyzed the high regioselective (98 %) C-H hydroxylation of omeprazole sulfide to 5'-OH omeprazole sulfide with a high conversion yield (85-90 %). CONCLUSIONS: A highly efficient synthesis of 5'-OH omeprazole sulfide was developed using CYP102A1 from Bacillus megaterium as a biocatalyst.

Structural and biophysical characterization of human cytochromes P450 2B6 and 2A6 bound to volatile hydrocarbons: analysis and comparison.

X-ray crystal structures of complexes of cytochromes CYP2B6 and CYP2A6 with the monoterpene sabinene revealed two distinct binding modes in the active sites. In CYP2B6, sabinene positioned itself with the putative oxidation site located closer to the heme iron. In contrast, sabinene was found in an alternate conformation in the more compact CYP2A6, where the larger hydrophobic side chains resulted in a significantly reduced active-site cavity. Furthermore, results from isothermal titration calorimetry indicated a much more substantial contribution of favorable enthalpy to sabinene binding to CYP2B6 as opposed to CYP2A6, consistent with the previous observations with (+)-α-pinene. Structural analysis of CYP2B6 complexes with sabinene and the structurally similar (3)-carene and comparison with previously solved structures revealed how the movement of the F206 side chain influences the volume of the binding pocket. In addition, retrospective analysis of prior structures revealed that ligands containing -Cl and -NH functional groups adopted a distinct orientation in the CYP2B active site compared with other ligands. This binding mode may reflect the formation of Cl-π or NH-π bonds with aromatic rings in the active site, which serve as important contributors to protein-ligand binding affinity and specificity. Overall, the findings from multiple techniques illustrate how drugs metabolizing CYP2B6 and CYP2A6 handle a common hydrocarbon found in the environment. The study also provides insight into the role of specific functional groups of the ligand that may influence the binding to CYP2B6.

Functional importance of a peripheral pocket in mammalian cytochrome P450 2B enzymes.

The functional importance of a peripheral pocket found in previously published X-ray crystal structures of CYP2B4 and CYP2B6 was probed using a biophysical approach. Introduction of tryptophan within the pocket of CYP2B4 at F202 or I241 leads to marked impairment of 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) or 7-benzyloxyresorufin O-dealkylation efficiency; a similar substitution at F195, near the surface access to the pocket, does not affect these activities. The analogous CYP2B6 F202W mutant is inactive in the 7-EFC O-dealkylation assay. The stoichiometry of 7-EFC deethylation suggested that the decreased activity of F202W and I241W in CYP2B4 and lack of activity of F202W in CYP2B6 coincided with a sharp increase in the flux of reducing equivalents through the oxidase shunt to produce excess water. The results indicate that the chemical identity of residues within this peripheral pocket, but not at the mouth of the pocket, is important in substrate turnover and redox coupling, likely through effects on active site topology.

Impact of lysophosphatidylcholine on survival and function of UEA-1(+)acLDL (+) endothelial progenitor cells in patients with coronary artery disease.

Lysophosphatidylcholine (LPC) generated from oxidized low-density lipoprotein by lipoprotein-associated phospholipase A2 plays a key role in plaque inflammation and vulnerability. Endothelial progenitor cells (EPCs) can repair injured endothelium and exert anti-inflammatory effects of vulnerable plaque. We study the impact and mechanisms of LPC on UEA-1 and acLDL binding EPCs (UEA-1(+)acLDL(+) EPCs). UEA-1(+)acLDL(+) EPCs from coronary artery disease (CAD) patients were cultured and exposed to LPC at different concentrations and different timepoints. We determined the significant concentration (40 µM). UEA-1(+)acLDL(+) EPCs were preincubated for 30 min with pravastatin (20 µM) with LY249002, a specific inhibitor of the Akt signaling pathway, and exposed for 24 h to LPC 40 µM. The survival, migration, adhesion, and proliferation of UEA-1(+)acLDL(+) EPCs were assessed. To examine the mechanisms of LPC toxicity and pravastatin effects, phosphorylated Akt and endothelial nitric oxide synthase (eNOS) levels and the ratio of Bcl-2/Bax protein expression were assessed. LPC induced apoptosis and impaired migration and adhesion of UEA-1(+)acLDL(+) EPCs significantly. The detrimental effects of LPC were attenuated by pravastatin. However, when UEA-1(+)acLDL(+) EPCs were pretreated with pravastatin and LY249002, a specific inhibitor of the Akt signaling pathway, simultaneously, the beneficial effects of pravastatin were abolished. Furthermore, LPC suppressed Akt and eNOS phosphorylation and increased Bcl-2/Bax expression. The effects of LPC on Akt/eNOS and Bcl-2/Bax activity were reversed by pravastatin. In conclusion, LPC inhibited UEA-1(+)acLDL(+) EPCs survival and impaired its functions, and these were attributable to inhibition of the Akt/eNOS and Bcl-2/Bax pathway. Pravastatin reversed the detrimental action of LPC. These findings suggest that LPC inhibition can be a possible strategy for CAD through EPC revitalization.

Effects of electroacupuncture on endothelial function and circulating endothelial progenitor cells in patients with cerebral infarction.

This study evaluated the effects of electroacupuncture (EA) on endothelial function and endothelial progenitor cells (EPC) in patients with cerebral infarction. In a randomized, placebo-controlled, crossover study, 20 patients with cerebral infarction were randomized into two treatment groups: EA or placebo. Before and after each intervention, pulse amplitude tonometry (PAT) was used to assess endothelial function and peripheral blood was analyzed for the number of EPCs. Circulating EPCs were quantified by flow cytometry as CD45(low) CD34(+) KDR2(+) cells. Plasma vascular endothelial growth factor (VEGF) and interleukin (IL)-10 levels were measured. Seven days later, crossover was performed on each group, with each group receiving the other treatment using the same protocol. The PAT hyperemia ratio ranged from 1.57 ± 0.41 to 2.04 ± 0.51 after EA, representing a significant improvement (P = 0.002); however, there was no improvement in the placebo group (P = 0.48). Circulating EPCs, as measured by flow cytometry, increased to 110.6 ± 74.3/100 µL in the EA group (P = 0.001) but did not change in the placebo group (45.9 ± 35.3/100 µL, P = 0.08). The increases in the number of EPCs and the PAT ratio after treatment were correlated (r = 0.78, P < 0.001). Plasma VEGF levels increased with EA compared to baseline (261.2 ± 34.0 vs 334.9 ± 80.5 pg/mL, P = 0.003). The number of circulating EPCs was positively correlated with plasma levels of VEGF (r = 0.50, P = 0.02). In conclusion, EA induced improvement of EPC levels and the PAT ratio in patients with cerebral infarction.

Functional characterization of cytochromes P450 2B from the desert woodrat Neotoma lepida.

Mammalian detoxification processes have been the focus of intense research, but little is known about how wild herbivores process plant secondary compounds, many of which have medicinal value or are drugs. cDNA sequences that code for three enzymes of the cytochrome P450 (CYP) 2B subfamily, here termed 2B35, 2B36, and 2B37 have been recently identified from a wild rodent, the desert woodrat (Malenke et al., 2012). Two variant clones of each enzyme were engineered to increase protein solubility and to facilitate purification, as reported for CYP2B enzymes from multiple species. When expressed in Escherichia coli each of the woodrat proteins gave the characteristic maximum at 450nm in a reduced carbon monoxide difference spectrum but generally expressed at lower levels than rat CYP2B1. Two enzymes, 2B36 and 2B37, showed dealkylation activity with the model substrates 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin, whereas 2B35 was inactive. Binding of the monoterpene (+)-α-pinene produced a Type I shift in the absorbance spectrum of each enzyme. Mutation of 2B37 at residues 114, 262, or 480, key residues governing ligand interactions with other CYP2B enzymes, did not significantly change expression levels or produce the expected functional changes. In summary, two catalytic and one ligand-binding assay are sufficient to distinguish among CYP2B35, 2B36, and 2B37. Differences in functional profiles between 2B36 and 2B37 are partially explained by changes in substrate recognition site residue 114, but not 480. The results advance our understanding of the mechanisms of detoxification in wild mammalian herbivores and highlight the complexity of this system.

The role of cytochrome P450 2B6 and 2B4 substrate access channel residues predicted based on crystal structures of the amlodipine complexes.

Recent X-ray crystal structures of human cytochrome P450 2B6 and rabbit cytochrome P450 2B4 in complex with amlodipine showed two bound ligand molecules, one in the active site and one in the substrate access channel. Based on the X-ray crystal structures, we investigated the interactions of P450 2B4 and 2B6 with amlodipine using absorbance spectroscopy, and determined the steady-state kinetics of 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin oxidation by some access channel mutants to evaluate the functional role of these residues in substrate turnover. The results of absorbance titrations are consistent with a simple mechanism with two parallel binding events that result in the formation of the enzyme complex with two molecules of amlodipine. Using this model we were able to resolve two separate ligand-binding events, which are characterized by two distinct KD values in each enzyme. The access channel mutants R73K in P450 2B6 and R73K, V216W, L219W, and F220W in P450 2B4 showed a significant decrease in kcat/KM with the both substrates. Overall, the results suggest that P450 2B4 and 2B6 form an enzyme complex with two molecules of amlodipine in solution, and R73, V216, L219 and F220 in P450 2B4 may play an important role in substrate metabolism.

Effects of sildenafil on nanostructural and nanomechanical changes in mitochondria in an ischaemia-reperfusion rat model.

Sildenafil exerts cardioprotective effects by activating the opening of mitochondrial ATP-sensitive potassium channels to attenuate ischaemia-reperfusion (IR) injury. In the present study, we used atomic force microscopy (AFM) to investigate changes in mitochondrial morphology and properties to assess sildenafil-mediated cardioprotection in a rat myocardial infarction model. To investigate the cardioprotective effects of sildenafil, we used an in vivo Sprague-Dawley rat model of IR. Rats were randomly divided into three groups: (i) sham-operated rats (control; n = 5); (ii) IR-injured rats treated with vehicle (normal saline; IR; n = 10); and (iii) IR-injured rats treated with 0.75 mg/kg, i.p., sildenafil (IR + Sil; n = 10). Morphological and mechanical changes to mitochondria were analysed by AFM. Infarct areas were significantly reduced in sildenafil-treated rats (7.8 ± 3.9% vs 20.4 ± 7.0% in the sildenafil-treated and untreated IR groups, respectively; relative reduction 62%; P < 0.001). Analysis of mitochondria by AFM showed that IR injury significantly increased the areas of isolated mitochondria compared with control (24 150 ± 18 289 vs 1495 ± 1139 nm(2) , respectively; P < 0.001), indicative of mitochondrial swelling. Pretreatment with sildenafil before IR injury reduced the mitochondrial areas (7428 ± 3682 nm(2) ; P < 0.001; relative reduction 69.2% compared with the IR group) and ameliorated the adhesion force of mitochondrial surfaces. Together, these results suggest that sildenafil has cardioprotective effects against IR injury in a rat model by improving the morphological and mechanical characteristics of mitochondria.

Estrogen-related receptor γ controls hepatic CB1 receptor-mediated CYP2E1 expression and oxidative liver injury by alcohol.

BACKGROUND: The hepatic endocannabinoid system and cytochrome P450 2E1 (CYP2E1), a key enzyme causing alcohol-induced reactive oxygen species (ROS) generation, are major contributors to the pathogenesis of alcoholic liver disease. The nuclear hormone receptor oestrogen-related receptor γ (ERRγ) is a constitutively active transcriptional activator regulating gene expression. OBJECTIVE: To investigate the role of ERRγ in the alcohol-mediated regulation of CYP2E1 and to examine the possibility to control alcohol-mediated oxidative stress and liver injury through an ERRγ inverse agonist. DESIGN: For chronic alcoholic hepatosteatosis study, C57BL/6J wild-type and CB1(-/-) mice were administered alcohol for 4 weeks. GSK5182 and chlormethiazole (CMZ) were given by oral gavage for the last 2 weeks of alcohol feeding. Gene expression profiles and biochemical assays were performed using the liver or blood of mice. RESULTS: Hepatic ERRγ gene expression induced by alcohol-mediated activation of CB1 receptor results in induction of CYP2E1, while liver-specific ablation of ERRγ gene expression blocks alcohol-induced expression of CYP2E1 in mouse liver. An ERRγ inverse agonist significantly ameliorates chronic alcohol-induced liver injury in mice through inhibition of CYP2E1-mediated generation of ROS, while inhibition of CYP2E1 by CMZ abrogates the beneficial effects of the inverse agonist. Finally, chronic alcohol-mediated ERRγ and CYP2E1 gene expression, ROS generation and liver injury in normal mice were nearly abolished in CB1(-/-) mice. CONCLUSIONS: ERRγ, as a previously unrecognised transcriptional regulator of hepatic CB1 receptor, controls alcohol-induced oxidative stress and liver injury through CYP2E1 induction, and its inverse agonist could ameliorate oxidative liver injury due to chronic alcohol exposure.

The effectiveness of tumor necrosis factor-α blocker therapy in patients with axial spondyloarthritis who failed conventional treatment: a comparative study focused on improvement in ASAS Health Index.

Objective: The purpose of this study is to evaluate the impact of tumor necrosis factor (TNF)-α blocker therapy on the Assessment of SpondyloArthritis international Society Health Index (ASAS-HI) among patients who have failed conventional nonsteroidal anti-inflammatory drugs. Methods: A comparative study was conducted involving axial spondyloarthritis (axSpA) patients treated with either TNF-α blocker or conventional therapy. Patient data, including demographics, disease characteristics, and ASAS-HI scores, were collected before and after treatment. Statistical analysis was performed to compare changes in ASAS-HI scores between the TNF-α blocker and the conventional therapy group. Results: The study population consisted of patients with axSpA, with a mean age of 38.3 years in conventional treatment group and 29.3 years in TNF-α blocker group. Most variables, including C-reactive protein levels, other comorbidities, and disease assessment scores showed no significant difference between groups. Longitudinal analysis within each treatment group from Week 0 to 12 showed no significant change in the conventional treatment group, whereas the TNF-α blocker group experienced a significant reduction in ASAS-HI scores, demonstrating the effectiveness of the treatment. The TNF-α blocker group exhibited a significantly greater improvement in ASAS-HI scores compared to the conventional therapy group. The Bath Ankylosing Spondylitis Functional Index and the Bath Ankylosing Spondylitis Disease Activity Index demonstrated strong positive correlations with ASAS-HI scores, indicating higher disease activity and functional limitation are associated with worse health outcomes in patients. Conclusion: The research demonstrates that ASAS-HI scores significantly improve with TNF-α blocker therapy in axSpA patients, underscoring ASAS-HI's effectiveness as a tool for evaluating drug responses.

Exosome-mediated delivery of super-repressor IκBα alleviates inflammation and joint damages in rheumatoid arthritis.

BACKGROUND: This study aims to investigate the potential anti-inflammatory effects of exosomes engineered to carry super-repressor IκB (Exo-srIκB), an exosome-based NF-κB inhibitor, in the context of RA. METHODS: Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were collected from patients diagnosed with RA and treated with Exo-srIκB to test the therapeutic potential. Flow cytometry analysis was performed to assess the production of inflammatory cytokines (IL-17A and GM-CSF) by the cells. ELISA was utilized to measure the levels of TNF-α, IL-17A, IL-6, and GM-CSF. Arthritis was induced in SKG mice by intraperitoneal injection of curdlan. DBA/1 J mice were used in collagen-induced arthritis (CIA) experiments. After the development of arthritis, mice were injected with either Exo-Naïve (control exosome) or Exo-srIκB. Arthritis scores were recorded biweekly, and histological observations of the ankle joint were conducted using H&E and safranin-O staining. Additionally, bone erosion was evaluated using micro-CT imaging. RESULTS: In the ex vivo study involving human PBMCs and SFMCs, treatment with Exo-srIκB demonstrated a notable reduction in inflammatory cytokines. Furthermore, in both the SKG and CIA models, Exo-srIκB treatment exhibited significant reductions in inflammation, cartilage destruction, and bone erosion within the joint tissues when compared to the Exo-Naive control group. Additionally, the radiographic score assessed through microCT showed a significant decrease compared to the Exo-Naive control group. CONCLUSION: Overall, these findings suggest that Exo-srIκB possesses anti-inflammatory properties in human RA cells and animal models, making it a promising therapeutic candidate for the treatment of RA.

Structural and thermodynamic basis of (+)-α-pinene binding to human cytochrome P450 2B6.

Despite recent advances in atomic-level understanding of drug and inhibitor interactions with human cytochromes P450, the decades-old questions of chemical and structural determinants of hydrocarbon binding are still unanswered. (+)-α-Pinene is a monoterpene hydrocarbon that is widely distributed in the environment and a potent P450 2B inhibitor. Therefore, a combined biophysical and structural analysis of human P450 2B6 interactions with (+)-α-pinene was undertaken to elucidate the basis of the very high affinity binding. Binding of (+)-α-pinene to the P450 active site was demonstrated by a Type I spectral shift. Thermodynamics of ligand binding were explored using isothermal titration calorimetry and compared to those of P450 2A6, which is much less flexible than 2B6 based on comparison of multiple X-ray crystal structures. Consistent with expectation, entropy is the major driving force for hydrocarbon binding to P450 2A6, as evidenced by the calorimetric results. However, formation of the 2B6-(+)-α-pinene complex has a significant enthalpic component. A 2.0 Å resolution crystal structure of this enzyme-ligand complex reveals that the highly plastic 2B6 utilizes previously unrecognized rearrangements of protein motifs. The results indicate that the specific components of enthalpic contribution to ligand binding are closely tied to the degree of enzyme flexibility.

X-ray crystal structure of the cytochrome P450 2B4 active site mutant F297A in complex with clopidogrel: insights into compensatory rearrangements of the binding pocket.

Prior X-ray crystal structures of cytochrome P450 2B4 revealed the pivotal role of rearrangement of the side chains of residues F206 and F297 in the active site in accommodating various inhibitors or substrates. To explore the role of these residues, 2B4 F206A and F297A were created by site-directed mutagenesis and characterized functionally. The structure of F297A with clopidogrel demonstrated the reorientation of the ligand such that the methyl ester group is oriented toward the heme, whereas the thiophene moiety now extends to the additional void in the F297A mutant. Most interestingly, movement of the I helix and several amino acid side chains within the active site was observed in apparent response to the altered binding orientation. Results of flexible docking using the 2B4 wild type or the F297A-virtual mutant positioned either the thiophene or chlorophenyl group closer to heme. However, docking of clopidogrel using the real F297A mutant or a virtual mutant with the I-helix re-positioned oriented clopidogrel preferentially with either the methyl ester or the chlorophenyl group closest to heme. The study provides insight into how the altered active site adapts to accommodate and interact with the substrate in a distinct orientation while maintaining the overall closed protein conformation.

A Potential Predictive Role of the Scalp Microbiome Profiling in Patients with Alopecia Areata: Staphylococcus caprae, Corynebacterium, and Cutibacterium Species.

Little is known about the scalp bacterial composition of alopecia areata (AA) patients. The aim of this study was to investigate the differences in the scalp microbiome of AA patients according to their prognosis, in addition to healthy controls. A total of 33 AA patients and 12 healthy controls (HC) were included in this study. The microbiomes were characterized by sequencing 16S rRNA genes on the Illumina MiSeq platform. The scalp microbiome was more diverse in AA patients compared to HC, but not significantly different according to the severity of AA. Nevertheless, the higher proportion of Corynebacterium species and the lower proportion of Staphylococcus caprae among the Staphylococcus species were noticed in severe AA patients compared to HC or mild AA. The higher ratio of Cutibacterium species to S. caprae was noticed in severe AA. We highlight the potential predictive role of scalp microbiome profiling to a worse prognosis of patients with alopecia areata.

Breast Tumor Cell-Stimulated Bone Marrow-Derived Mesenchymal Stem Cells Promote the Sprouting Capacity of Endothelial Cells by Promoting VEGF Expression, Mediated in Part through HIF-1α Increase.

Breast tumor cells recruit bone marrow-derived mesenchymal stem cells (BM-MSCs) and alter their cellular characteristics to establish a tumor microenvironment. BM-MSCs enhance tumor angiogenesis through various mechanisms. We investigated the mechanisms by which BM-MSCs promote angiogenesis in response to breast tumor. Conditioned media from MDA-MB-231 (MDA CM) and MCF7 (MCF7 CM) breast tumor cells were used to mimic breast tumor conditions. An in vitro spheroid sprouting assay using human umbilical vein endothelial cells (HUVECs) was conducted to assess the angiogenesis-stimulating potential of BM-MSCs in response to breast tumors. The ROS inhibitor N-acetylcysteine (NAC) and JAK inhibitor ruxolitinib attenuated increased HIF-1α in BM-MSCs in response to MDA CM and MCF7 CM. HIF-1α knockdown or HIF-1ß only partially downregulated VEGF expression and, therefore, the sprouting capacity of HUVECs in response to conditioned media from BM-MSCs treated with MDA CM or MCF7 CM. Inactivation of the VEGF receptor using sorafenib completely inhibited the HUVECs' sprouting. Our results suggest that increased HIF-1α expression under normoxia in BM-MSCs in response to breast tumor cells is mediated by ROS and JAK/Stat3, and that both HIF-1α-dependent and -independent mechanisms increase VEGF expression in BM-MSCs to promote the angiogenic sprouting capacity of endothelial cells in a VEGF-dependent manner.

Beta sheet 2-alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners.

As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein-protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1*CPR complex and inactive (CYP2B1)(2)*CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge-charge interactions between CPR and complex partners.

Functional and conformational modulation of human cytochrome P450 1B1 by anionic phospholipids.

We investigated the interaction of human P450 1B1 (CYP1B1) with various phospholipid bilayers using the N-terminally deleted (Delta2-4)CYP1B1 and (Delta2-26)CYP1B1 enzymes. Among anionic phospholipids, phosphatidic acid (PA) and cardiolipin specifically increased the catalytic activities, membrane binding affinities, and thermal stabilities of both CYP1B1 proteins when phosphatidylcholine matrix was gradually replaced with these anionic phospholipids. PA- or cardiolipin-dependent changes of CYP1B1 conformation were revealed by altered Trp fluorescence and CD spectra. However, both PA and cardiolipin exerted more significant effects with the (Delta2-4)CYP1B1 than the (Delta2-26)CYP1B1 implying the functional importance of N-terminal region for the interaction with the phospholipid membranes. In contrast, other anionic phospholipids such as phosphatidylserine and the neutral phospholipid phosphatidylethanolamine had no apparent effects on the catalytic activity or conformation of CYP1B1. These data suggest that the chemical and physical properties of membranes influenced by PA or cardiolipin composition are critical for the functional roles of CYP1B1.