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苏木精 certified by the Biological Stain Commission; CAS Number: 517-28-2; EC Number: 208-237-3; Synonyms: 苏木精; find Sigma-Aldrich-H3136 MSDS, related peer-reviewed papers, technical documents, similar products & more at Sigma-Aldrich

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苏木精

Expression of matrilin-2 in liver cirrhosis and hepatocellular carcinoma.

S-nitrosylation of fatty acid synthase regulates its activity through dimerization.

Histologic validation of locus coeruleus MRI contrast in post-mortem tissue.

Expression of matrilin-2 in liver cirrhosis and hepatocellular carcinoma

S-nitrosylation of fatty acid synthase regulates its activity through dimerization

Lung Section Staining and Microscopy

Although an important biomarker in breast cancer, Ki67 lacks scoring standardization, which has limited its clinical use. Our previous study found variability when laboratories used their own scoring methods on centrally stained tissue microarray slides. In this current study, 16 laboratories from eight countries calibrated to a specific Ki67 scoring method and then scored 50 centrally MIB-1 stained tissue microarray cases. Simple instructions prescribed scoring pattern and staining thresholds for determination of the percentage of stained tumor cells. To calibrate, laboratories scored 18 'training' and 'test' web-based images. Software tracked object selection and scoring. Success for the calibration was prespecified as Root Mean Square Error of scores compared with reference <0.6 and Maximum Absolute Deviation from reference <1.0 (log2-transformed data). Prespecified success criteria for tissue microarray scoring required intraclass correlation significantly >0.70 but aiming for observed intraclass correlation ≥0.90. Laboratory performance showed non-significant but promising trends of improvement through the calibration exercise (mean Root Mean Square Error decreased from 0.6 to 0.4, Maximum Absolute Deviation from 1.6 to 0.9; paired t-test: P=0.07 for Root Mean Square Error, 0.06 for Maximum Absolute Deviation). For tissue microarray scoring, the intraclass correlation estimate was 0.94 (95% credible interval: 0.90-0.97), markedly and significantly >0.70, the prespecified minimum target for success. Some discrepancies persisted, including around clinically relevant cutoffs. After calibrating to a common scoring method via a web-based tool, laboratories can achieve high inter-laboratory reproducibility in Ki67 scoring on centrally stained tissue microarray slides. Although these data are potentially encouraging, suggesting that it may be possible to standardize scoring of Ki67 among pathology laboratories, clinically important discrepancies persist. Before this biomarker could be recommended for clinical use, future research will need to extend this approach to biopsies and whole sections, account for staining variability, and link to outcomes.

An international study to increase concordance in Ki67 scoring.

NO regulates a variety of physiological processes, including cell proliferation, differentiation, and inflammation. S-nitrosylation, a NO-mediated reversible protein modification, leads to changes in the activity and function of proteins. In particular, the role of S-nitrosylation during adipogenesis is largely unknown. We hypothesized that the normal physiological levels of NO, but not the excess levels generated under severe conditions, such as inflammation, may be critically involved in the proper regulation of adipogenesis. We found that endogenous S-nitrosylation of proteins was required for adipocyte differentiation. By performing a biotin-switch assay, we identified FAS, a key lipogenic enzyme in adipocytes, as a target of S-nitrosylation during adipogenesis. Interestingly, we also observed that the dimerization of FAS increased in parallel with the amount of S-nitrosylated FAS during adipogenesis. In addition, we found that exogenous NO enhanced the dimerization and the enzymatic activity of FAS. Moreover, site-directed mutagenesis of three predicted S-nitrosylation sites indicated that S-nitrosylation of FAS at Cys(1471)and Cys(2091), but not at Cys(1127), increased its enzymatic activity. Taken together, these results suggest that the S-nitrosylation of FAS at normal physiological levels of NO increases its activity through dimerization and may contribute to the proper regulation of adipogenesis.

Our aim was to investigate the pertinence of diffusion and perfusion magnetic resonance imaging (MRI) parameters obtained at 17.2 T in a 9L glioma rat brain tumor model to evaluate tumor tissue characteristics. The local animal ethics advisory committee approved this study. 9L glioma cells were injected intracerebrally to 14 Fischer rats. The animals were imaged at 7 or 12 days after implantation on a 17.2-T MRI scanner, using 72 different b values (2-3025 s/mm(2)). The signal attenuation, S/So, was fitted using a kurtosis diffusion model (ADCo and K) and a biexponential diffusion model (fractions ffast and fslow and diffusion coefficients Dfast and Dslow) using b values greater than 300 s/mm(2). To bridge the 2 models, an average diffusion coefficient <D> and a biexponential index were estimated from the biexponential model as ADCo and K equivalents, respectively. Intravoxel incoherent motion perfusion-related parameters were obtained from the residual signal at low b values, after the diffusion component has been removed. Diffusion and perfusion maps were generated for each fitted parameter on a pixel-by-pixel basis, and regions of interest were drawn in the tumor and contralateral side to retrieve diffusion and perfusion parameters. All rats were killed and cellularity and vascularity were quantitatively assessed using histology for comparison with diffusion and perfusion parameters. Intravoxel incoherent motion maps clearly highlighted tumor areas as generally heterogeneous, as confirmed by histology. For diffusion parameters, ADCo and <D> were not significantly different between the tumor and contralateral side, whereas K in the tumor was significantly higher than in contralateral basal ganglia (P < 0.0001), as well as biexponential index (P < 0.001). ADCo and <D> in the tumor at day 7 were significantly higher than at day 12 (P < 0.01 and P < 0.001, respectively). fIVIM in the tumor from the kurtosis diffusion model was significantly higher than in contralateral basal ganglia (P < 0.001). fIVIM in the tumor at day 7 was significantly higher than in the tumor at day 12 (P < 0.0001). There was no significant difference for D* between the tumor and contralateral side (P = 0.06). A significant negative correlation was found between tumor vascularity and fIVIM (P < 0.05) as well as between tumor cell count and <D> (P < 0.01). Quantitative non-Gaussian diffusion and perfusion MRI can provide valuable information on microvasculature and tissue structure to improve characterization of brain tumors.

Characterization of glioma microcirculation and tissue features using intravoxel incoherent motion magnetic resonance imaging in a rat brain model.

Uveal melanoma is a lethal cancer with a strong propensity to metastasize. Limited therapeutic options are available once the disease has disseminated. A strong predictor for metastasis is the loss of chromosome 3. Inactivating mutations in BAP1 encoding the BRCA1-associated protein 1 and located on chromosome 3p21.1, have been described in uveal melanoma and other types of cancer. In this study, we determined the prevalence of somatic BAP1 mutations and examined whether these mutations correlate with the functional expression of BAP1 in uveal melanoma tissue and with other clinical, histopathological and chromosomal parameters. We screened a cohort of 74 uveal melanomas for BAP1 mutations, using different deep sequencing methods. The frequency of BAP1 mutations in our study group was 47%. The expression of BAP1 protein was studied using immunohistochemistry. BAP1 staining was absent in 43% of the cases. BAP1 mutation status was strongly associated with BAP1 protein expression (P<0.001), loss of chromosome 3 (P<0.001), and other aggressive prognostic factors. Patients with a BAP1 mutation and absent BAP1 expression had an almost eightfold higher chance of developing metastases compared with those without these changes (P=0.002). We found a strong correlation between the immunohistochemical and sequencing data and therefore propose that, immunohistochemical screening for BAP1 should become routine in the histopathological work-up of uveal melanoma. Furthermore, our analysis indicates that loss of BAP1 may be particularly involved in the progression of uveal melanoma to an aggressive, metastatic phenotype.

Clinical significance of immunohistochemistry for detection of BAP1 mutations in uveal melanoma.

Cancer progression involves a rare population of undifferentiated cancer-initiating cells that have stem cell-like properties for self-renewal capacity and high tumorigenicity. We investigated how maintenance of pancreatic cancer-initiating cells is influenced by Smad4, which is frequently deleted or mutated in pancreatic cancers cells. Smad4 silencing up-regulated the expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA, whereas forced expression of Smad4 in pancreatic cancer cells down-regulated it. Smad4 and ALDH1 expression inversely correlated in some human clinical pancreatic adenocarcinoma tissues, suggesting that ALDH1 in pancreatic cancer cells was associated with decreased Smad4 expression. We then examined whether ALDH1 served as a marker of pancreatic cancer-initiating cells. Pancreatic cancer cells contained ALDH1(hi) cells in 3% to 10% of total cells, with high tumorigenic potential. Because Smad4 is a major mediator of transforming growth factor (TGF)-β family signaling, we investigated the regulatory mechanism of ALDH activity by TGF-β and bone morphogenetic proteins. Treatment with TGF-β attenuated ALDH1(hi) cells in several pancreatic cancer cells, whereas bone morphogenetic protein-4 was not as potent. Biochemical experiments revealed that TGF-β regulated ALDH1A1 mRNA transcription through binding of Smad4 to its regulatory sequence. It appears that TGF-β negatively regulates ALDH1 expression in pancreatic cancer cells in a Smad-dependent manner and in turn impairs the activity of pancreatic cancer-initiating cells.

Smad4 Decreases the Population of Pancreatic Cancer-Initiating Cells through Transcriptional Repression of ALDH1A1.

Medulloblastoma is the most common malignant brain tumor in children, but the cells from which it arises remain unclear. Here we examine the origin of medulloblastoma resulting from mutations in the Sonic hedgehog (Shh) pathway. We show that activation of Shh signaling in neuronal progenitors causes medulloblastoma by 3 months of age. Shh pathway activation in stem cells promotes stem cell proliferation but only causes tumors after commitment to-and expansion of-the neuronal lineage. Notably, tumors initiated in stem cells develop more rapidly than those initiated in progenitors, with all animals succumbing by 3-4 weeks. These studies suggest that medulloblastoma can be initiated in progenitors or stem cells but that Shh-induced tumorigenesis is associated with neuronal lineage commitment.

Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells.

Patients with diabetes have an increased risk of developing atherosclerosis. Endothelial dysfunction, characterized by the lowered bioavailability of endothelial NO synthase (eNOS)-derived NO, is a critical inducer of atherosclerosis. However, the protective aspect of eNOS in diabetes-associated atherosclerosis remains controversial, a likely consequence of its capacity to release both protective NO or deleterious oxygen radicals in normal and disease settings, respectively. Harnessing the atheroprotective activity of eNOS in diabetic settings remains elusive, in part due to the lack of endogenous eNOS-specific NO release activators. We have recently shown in vitro that eNOS-derived NO release can be increased by blocking its binding to Caveolin-1, the main coat protein of caveolae, using a highly specific peptide, CavNOxin. However, whether targeting eNOS using this peptide can attenuate diabetes-associated atherosclerosis is unknown. In this study, we show that CavNOxin can attenuate atherosclerotic burden by ∼84% in vivo. In contrast, mice lacking eNOS show resistance to CavNOxin treatment, indicating eNOS specificity. Mechanistically, CavNOxin lowered oxidative stress markers, inhibited the expression of proatherogenic mediators, and blocked leukocyte-endothelial interactions. These data are the first to show that endogenous eNOS activation can provide atheroprotection in diabetes and suggest that CavNOxin is a viable strategy for the development of antiatherosclerotic compounds.

Direct Endothelial Nitric Oxide Synthase Activation Provides Atheroprotection in Diabetes-Accelerated Atherosclerosis.

Estrogen receptor β (ERβ) is emerging as a critical factor in understanding prostate cancer biology. Although reduced in prostate cancer above Gleason grade 3, ERβ is a potential drug target at the initial stage of the disease. In human prostate cancer cells, we found that ERβ causes apoptosis by increasing the expression of pro-apoptotic factor p53-upregulated modulator of apoptosis (PUMA), independent of p53, but dependent on the forkhead transcription factor class-O family member, FOXO3a. FOXO3a has previously been shown to induce PUMA after growth factor withdrawal and inhibition of the Akt pathway. Surprisingly, the phosphorylation of FOXO3a remained unchanged, while the mRNA and total protein levels of FOXO3a were increased in response to ERβ expression or treatment of PC3, 22Rv1 and LNCaP cells with the ERβ-specific ligands 3β-Adiol (5α-androstane-3β,17β-diol), DPN (diarylpropionitrile) or 8β-VE2 (8-vinylestra-1,3,5 (10)-triene-3,17β-diol). Knockdown of FOXO3a or ERβ expression abolished the increase of PUMA in response to 3β-Adiol in LNCaP and PC3 cells, suggesting that FOXO3a mediates the apoptotic effect of 3β-Adiol-activated ERβ. Moreover, the ventral prostate of ERβ-/- mice had decreased expression of FOXO3a and PUMA compared with the ERβ+/+ mice, indicating a relationship between ERβ and FOXO3a expression. The regulation of FOXO3a by ERβ in normal basal epithelial cells indicates a function of ERβ in cell differentiation and maintenance of cells in a quiescent state. In addition, the expression of ERβ, FOXO3a and PUMA is comparable and higher in benign prostatic hyperplasia than in prostate cancer Gleason grade 4 or higher, where there is substantial loss of ERβ, FOXO3a and PUMA. We conclude that ERβ induces apoptosis of prostate cancer cells by increasing transcription of FOXO3a, leading to an increase of PUMA and subsequent triggering of apoptosis via the intrinsic pathway involving caspase-9. Furthermore, we conclude that ligands specifically activating ERβ could be useful pharmaceuticals in the treatment of prostate cancer.

Estrogen receptor β upregulates FOXO3a and causes induction of apoptosis through PUMA in prostate cancer.

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (MSC) chondrogenesis. In this study, we combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in MSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce MSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

Tissue-engineered cartilage with inducible and tunable immunomodulatory properties.

Organs are generated from collections of cells that coalesce and remain together as they undergo a series of choreographed movements to give the organ its final shape. We know little about the cellular and molecular mechanisms that regulate tissue cohesion during morphogenesis. Extensive cell movements underlie eye development, starting with the eye field separating to form bilateral vesicles that go on to evaginate from the forebrain. What keeps eye cells together as they undergo morphogenesis and extensive proliferation is unknown. Here, we show that plexina2 (Plxna2), a member of a receptor family best known for its roles in axon and cell guidance, is required alongside the repellent semaphorin 6a (Sema6a) to keep cells integrated within the zebrafish eye vesicle epithelium. sema6a is expressed throughout the eye vesicle, whereas plxna2 is restricted to the ventral vesicle. Knockdown of Plxna2 or Sema6a results in a loss of vesicle integrity, with time-lapse microscopy showing that eye progenitors either fail to enter the evaginating vesicles or delaminate from the eye epithelium. Explant experiments, and rescue of eye vesicle integrity with simultaneous knockdown of sema6a and plxna2, point to an eye-autonomous requirement for Sema6a/Plxna2. We propose a novel, tissue-autonomous mechanism of organ cohesion, with neutralization of repulsion suggested as a means to promote interactions between cells within a tissue domain.

Sema6a and Plxna2 mediate spatially regulated repulsion within the developing eye to promote eye vesicle cohesion.

RAS and Rho small GTPases are key molecular switches that control cell dynamics, cell growth and tissue development through their distinct signaling pathways. Although much has been learnt about their individual functions in both cell and animal models, the physiological and pathophysiological consequences of their signaling crosstalk in multi-cellular context in vivo remain largely unknown, especially in liver development and liver tumorigenesis. Furthermore, the roles of RhoA in RAS-mediated transformation and their crosstalk in vitro remain highly controversial. When challenged with carcinogens, zebrafish developed liver cancer that resembles the human liver cancer both molecularly and histopathologically. Capitalizing on the growing importance and relevance of zebrafish (Danio rerio) as an alternate cancer model, we have generated liver-specific, Tet-on-inducible transgenic lines expressing oncogenic Kras(G12V), RhoA, constitutively active RhoA(G14V) or dominant-negative RhoA(T19N). Double-transgenic lines expressing Kras(G12V) with one of the three RhoA genes were also generated. Based on quantitative bioimaging and molecular markers for genetic and signaling aberrations, we showed that the induced expression of oncogenic Kras during early development led to liver enlargement and hepatocyte proliferation, associated with elevated Erk phosphorylation, activation of Akt2 and modulation of its two downstream targets, p21Cip and S6 kinase. Such an increase in liver size and Akt2 expression was augmented by dominant-negative RhoA(T19N), but was abrogated by the constitutive-active RhoA(G14V). Consequently, induced expression of the oncogenic Kras in adult transgenic fish led to the development of hepatocellular carcinomas. Survival studies further revealed that the co-expression of dominant-negative RhoA(T19N) with oncogenic Kras increased the mortality rate compared with the other single or double-transgenic lines. This study provides evidence of the previously unappreciated signaling crosstalk between Kras and RhoA in regulating liver overgrowth and liver tumorigenesis. Our results also implicate that activating Rho could be beneficial to suppress the Kras-induced liver malignancies.

Crosstalk of Ras and Rho: activation of RhoA abates Kras-induced liver tumorigenesis in transgenic zebrafish models.

Nonalcoholic fatty liver disease (NAFLD) encompasses a range of manifestations, including steatosis and cirrhosis. Progressive disease is characterized by hepatic leukocyte accumulation in the form of steatohepatitis. The adhesion molecule vascular adhesion protein-1 (VAP-1) is a membrane-bound amine oxidase that promotes leukocyte recruitment to the liver, and the soluble form (sVAP-1) accounts for most circulating monoamine oxidase activity, has insulin-like effects, and can initiate oxidative stress. Here, we determined that hepatic VAP-1 expression is increased in patients with chronic liver disease and that serum sVAP-1 levels are elevated in patients with NAFLD compared with those in control individuals. In 4 murine hepatic injury models, an absence or blockade of functional VAP-1 reduced inflammatory cell recruitment to the liver and attenuated fibrosis. Moreover, disease was reduced in animals expressing a catalytically inactive form of VAP-1, implicating enzyme activity in the disease pathogenesis. Within the liver, hepatic stromal cells expressed functional VAP-1, and evaluation of cultured cells revealed that sVAP-1 promotes leukocyte migration through catalytic generation of ROS, which depended on VAP-1 enzyme activity. VAP-1 enhanced stromal cell spreading and wound closure and modulated expression of profibrotic genes. Together, these results link the amine oxidase activity of VAP-1 with hepatic inflammation and fibrosis and suggest that targeting VAP-1 has therapeutic potential for NAFLD and other chronic fibrotic liver diseases.

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