T-LAK cell-originated protein kinase (TOPK) enhances androgen receptor splice variant (ARv7) and drives androgen-independent growth in prostate cancer.

Despite impressive advances in the treatment of prostate cancer with various efficacious inhibitors along the androgen/androgen receptor axis, eventual development of incurable metastatic Castration-Resistant Prostate Cancer (mCRPC) is inevitable and remains a major clinical challenge. Constitutively active androgen receptor (AR) spliced variants have emerged as primary means of resistance to anti-androgens and androgen synthesis inhibitors. The alternatively spliced AR variant, ARv7, has attracted significant interest due to its constitutively active status in CRPC that drives androgen-independence. Factors that are involved in regulating ARv7 levels in CRPC are not clearly known. We recently demonstrated that a protein kinase, T-LAK cell-originated protein kinase (TOPK) level correlates with the aggressiveness of prostate cancer and its invasive behavior. In this study, we investigated whether TOPK plays a role in driving androgen-independence in prostate cancer cells. Our data demonstrate that TOPK overexpression in androgen-dependent LNCaP and VCaP induces ARv7 and drives androgen-independent growth. On the other hand, pharmacological inhibition of TOPK in androgen-independent LNCaP95 and 22Rv1 represses AR transactivation, and AR stability. In summary, this study illustrates a direct role of TOPK in regulating ARv7 and driving androgen-independence in prostate cancer cells.

Biological significance of KRAS mutant allele expression in ovarian endometriosis.

KRAS is the most frequently mutated in ovarian endometriosis. However, it is unclear whether the KRAS mutant allele's mRNA is expressed and plays a biological role in ovarian endometriosis. Here, we performed mutation-specific RNA in situ hybridization to evaluate mutant allele expression of KRAS p.G12V, the most frequently detected mutation in ovarian endometriosis in our previous study, in formalin-fixed paraffin-embedded tissue (FFPE) samples of ovarian endometriosis, cancer cell lines, and ovarian cancers. First, we verified that mutant or wild-type allele of KRAS were expressed in all 5 cancer cell lines and 9 ovarian cancer cases corresponding to the mutation status. Next, we applied this assay to 26 ovarian endometriosis cases, and observed mutant allele expression of KRAS p.G12V in 10 cases. Mutant or wild-type allele of KRAS were expressed in line with mutation status in 12 available endometriosis cases for which KRAS gene sequence was determined. Comparison of clinical features between ovarian endometriosis with KRAS p.G12V mutant allele expression and with KRAS wild-type showed that KRAS p.G12V mutant allele expression was significantly associated with inflammation in ovarian endometriosis. Finally, we assessed the spatial distribution of KRAS mutant allele expression in 5 endometriosis cases by performing multiregional sampling. Intratumor heterogeneity of KRAS mutant allele expression was observed in two endometriosis cases, whereas the spatial distribution of KRAS p.G12V mutation signals were diffuse and homogenous in ovarian cancer. In conclusion, evaluation of oncogene mutant expression will be useful for clarifying the biological significance of oncogene mutations in benign tumors.

[Expression of TOPK/PBK in children with malignant lymphoma or reactive lymphoid hyperplasia].

OBJECTIVE: To study the difference in expression of TOPK/PBK in lymph nodes between children with malignant lymphoma and those with reactive lymphoid hyperplasia. METHODS: Eighty children with malignant lymphoma and twenty children with reactive lymphoid hyperplasia were enrolled as subjects. Immunohistochemistry was used to determine the expression of TOPK/PBK in all the subjects. The expression of TOPK/PBK was compared between the two groups. RESULTS: The TOPK/PBK-positivity rate was significantly higher in children with malignant lymphoma than in those with reactive lymphoid hyperplasia (P<0.05). There was no significant difference in the TOPK/PBK-positivity rate between the children with Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL). There were significant differences in the TOPK/PBK-positivity rate among children with different pathological types of NHL (P<0.05): the children with lymphoblastic lymphoma showed the highest TOPK/PBK-positivity rate and those with mature B-cell lymphoma and mature T/NK-cell lymphoma had a similar TOPK/PBK-positivity rate. CONCLUSIONS: The expression of TOPK/PBK is up-regulated in the lymph nodes of children with malignant lymphoma. The expression level of TOPK/PBK may be related to the pathological type of NHL.

Nano-LC-Q-TOF Analysis of Proteome Revealed Germination of Aspergillus flavus Conidia is Accompanied by MAPK Signalling and Cell Wall Modulation.

Aspergillus flavus is the second most leading cause of aspergillosis. The ability of A. flavus to adapt within the host environment is crtical for its colonization. Onset of germination of conidia is one of the crucial events; thus, in order to gain insight into A. flavus molecular adaptation while germination, protein profile of A. flavus was obtained. Approximately 82 % of conidia showed germination at 7 h; thus, samples were collected followed by protein extraction and subjected to nLC-Q-TOF mass spectrometer. Q-TOF data were analysed using Protein Lynx Global Services (PLGS 2.2.5) software. A total of 416 proteins were identified from UniProt Aspergillus species database. Orthologues of A. flavus was observed in A. fumigatus, A. niger, A. terreus, A. oryzae, etc. Proteins were further analysed in NCBI database, which showed that 27 proteins of A. flavus are not reported in UniProt and NCBI database. Functional characterization of proteins resulted majorly to cell wall synthesis and degradation, metabolisms (carbohydrate and amino acid metabolism), protein synthesis and degradation. Proteins/enzymes associated with aflatoxin biosynthesis were observed. We also observed Dicer-like proteins 1, 2 and autophagy-related proteins 2, 9, 18, 13, 11, 22. Expression of protein/enzymes associated with MAPK signalling pathway suggests their role during the germination process. Overall, the data present a catalogue of proteins/enzymes involved in the germination of A. flavus conidia and could also be applied to other Aspergillus species.

Systematic antibody generation and validation via tissue microarray technology leading to identification of a novel protein prognostic panel in breast cancer.

BACKGROUND: Although omic-based discovery approaches can provide powerful tools for biomarker identification, several reservations have been raised regarding the clinical applicability of gene expression studies, such as their prohibitive cost. However, the limited availability of antibodies is a key barrier to the development of a lower cost alternative, namely a discrete collection of immunohistochemistry (IHC)-based biomarkers. The aim of this study was to use a systematic approach to generate and screen affinity-purified, mono-specific antibodies targeting progression-related biomarkers, with a view towards developing a clinically applicable IHC-based prognostic biomarker panel for breast cancer. METHODS: We examined both in-house and publicly available breast cancer DNA microarray datasets relating to invasion and metastasis, thus identifying a cohort of candidate progression-associated biomarkers. Of these, 18 antibodies were released for extended analysis. Validated antibodies were screened against a tissue microarray (TMA) constructed from a cohort of consecutive breast cancer cases (n = 512) to test the immunohistochemical surrogate signature. RESULTS: Antibody screening revealed 3 candidate prognostic markers: the cell cycle regulator, Anillin (ANLN); the mitogen-activated protein kinase, PDZ-Binding Kinase (PBK); and the estrogen response gene, PDZ-Domain Containing 1 (PDZK1). Increased expression of ANLN and PBK was associated with poor prognosis, whilst increased expression of PDZK1 was associated with good prognosis. A 3-marker signature comprised of high PBK, high ANLN and low PDZK1 expression was associated with decreased recurrence-free survival (p < 0.001) and breast cancer-specific survival (BCSS) (p < 0.001). This novel signature was associated with high tumour grade (p < 0.001), positive nodal status (p = 0.029), ER-negativity (p = 0.006), Her2-positivity (p = 0.036) and high Ki67 status (p < 0.001). However, multivariate Cox regression demonstrated that the signature was not a significant predictor of BCSS (HR = 6.38; 95% CI = 0.79-51.26, p = 0.082). CONCLUSIONS: We have developed a comprehensive biomarker pathway that extends from discovery through to validation on a TMA platform. This proof-of-concept study has resulted in the identification of a novel 3-protein prognostic panel. Additional biochemical markers, interrogated using this high-throughput platform, may further augment the prognostic accuracy of this panel to a point that may allow implementation into routine clinical practice.

Anti-inflammatory effects of prosapogenin III from the dried roots of Liriope platyphylla in LPS-stimulated RAW264.7 cells.

Liriope platyphylla has been reported to possess various biological activities, including anti-asthma, anti-inflammation, anti-diabetes, and neuriotogenic properties. In this study, we evaluated the effects of prosapogenin III isolated from the roots of L. platyphylla (Liriopis Tuber) on inflammatory responses in lipopolysaccharide (LPS) stimulated RAW264.7 mouse macrophages. We investigated LPS-induced production/expression of inflammatory mediators such as nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxigenase-2 (COX-2), and proinflammatory cytokines, including interleukin-1ß (IL-1ß) and interleukin (IL)-6 in RAW264.7 cells. We also performed Western blot analysis for determination of the phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase (JNK), and nuclear translocation of nuclear factor-κB (NF-κB) in LPS-stimulated cells. Treatment with prosapogenin III resulted in significant inhibition of NO production in LPS-stimulated Raw264.7 cells through suppression of iNOS expression. Treatment with prosapogenin III resulted in a significant decrease in expressions of COX-2, IL-1ß, and IL-6 through down-regulation of their mRNA or protein in LPS-stimulated cells. In addition, treatment with prosapogenin III resulted in potently inhibited phosphorylation of three MAPKs, including ERK1/2, p38, and JNK in LPS-stimulated cells. Treatment with prosapogenin III also resulted in suppression of the nuclear translocation of NF-κB in LPS-stimulated cells. These results indicate that prosapogenin III of Liriopis Tuber has anti-inflammatory effects in activated macrophages through inhibition of production of inflammatory mediators by blockade of the MAPK/NF-κB pathway.

PTEN, Akt, MAPK, p53 and p95 expression to predict trastuzumab resistance in HER2 positive breast cancer.

PURPOSE: Mutations that activate the PIK3CA oncogene and inhibit the tumor suppressor gene PTEN action are commonly found in breast tumors. Akt is a key activator of cell survival. p53 is frequently found mutated in human tumors, and mutant p53 protein actively contributes to tumorigenesis. In selected cases of breast cancer, trastuzumab (TZMB) is incorporated in the primary treatment in the adjuvant and metastatic settings. Many studies have reported that selected patients are resistant to TZMB due to the presence of p95 HER2 fragments. To address this, we analysed PTEN, Akt, MAPK, p53 and p95 expression in breast cancer patients treated with TZMB. METHODS: Out of 90 patients histologically diagnosed with breast cancer between 2004 and 2011, analysed were 25 patients with HER2 positive, and estrogen (ER) and progesterone receptors (PR) negative, metastatic or locally advanced disease. All 25 patients were treated with TZMB and resistance to TZMB was assessed. All patients were on anthracycline-and taxane-containing regimens. Tissue samples were obtained from paraffin blocks and evaluated immunohistochemically for PTEN, Akt, MAPK, p53, and p95 expression. RESULTS: TZMB resistance was detected in 5 (20%) patients. Akt expression was positive in 2 patients (8%) and MAPK, p95, and p53 expression was positive in 1 patient (4%); PTEN expression was negative in 3 patients (12%). No significant differences were found between TZMB resistance and PTEN, Akt, MAPK, p53, and p95 expression. Subgroup analysis was carried out in the neoadjuvant treatment group. Complete pathologic response was detected in 3 patients (21.4%). Statistically significant differences were not found between the complete response rate and PTEN, Akt, MAPK, and p95 expression. There was a statistically significant correlation between p53 expression and complete pathologic response (p=0.02). CONCLUSION: No statistically significant correlation between TZMB resistance and the expression of these biomarkers was noted. In patients with HER2-positive breast cancer that were treated with 4 dose-dense sequential cycles of doxorubicin and cyclophosphamide, followed by TZMB and paclitaxel combination therapy in the neodjuvant setting, p53 expression could predict complete response to chemotherapy.

A continuous spectrophotometric assay for mitogen-activated protein kinase kinases.

We describe a convenient and simple continuous spectrophotometric method for the determination of mitogen-activated protein kinase (MAPK) kinase activity with its protein substrate. The assay relies on the measurement of phosphoprotein product generated in the first step of the MAPK kinase reaction. Dephosphorylation of the phosphoprotein is coupled to a MAPK phosphatase to generate phosphate, which is then used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methyl purine ribonucleoside. Of the reaction products ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360nm relative to the nucleoside and, hence, provides a spectrophotometric signal that can be continuously followed. In the presence of excess phosphatase, the phosphorylated protein substrate molecules undergo dephosphorylation almost immediately after their formation; the steady-state use of the resultant inorganic phosphate is a reflection of the constant initial velocity of the exchange reaction. The validity of this method has been confirmed by using it to measure the activities of MEK1 (MAPK/ERK kinase 1) and MKK6 (MAPK kinase 6) toward their physiological substrates. Our findings of the MAPK kinases in the current study provide evidence that the substrate binding affinities of this subfamily of protein kinases are at the submicromolar concentration.

Oligostilbenes from the seeds of Paeonia lactiflora as potent GLP-1 secretagogues targeting TGR5 receptor.

One unusual stilbene trimer-flavonoid hybrid, paeonilactiflobenoid (1), together with six known stilbenes (2-7) were isolated from the seeds of Paeonia lactiflora. The structure of 1 was elucidated with the aid of HRESIMS, 1D and 2D NMR, [α]D spectroscopic data and ECD calculation. Compounds 2-7 showed stimulative effects on GLP-1 secretion with promoting rates of 79.8%-880.4% (25 µM) and 217.6%-1089.4% (50 µM), more potent than the positive control, oleoylethanolamide (250.2% at 50 µM). Moreover, compounds 4 and 6 exhibited agonistic activity on the G protein-coupled receptor (GPCR) TGR5 with stimulative ratios of 40.2% and 40.5% at 50 µM, and 54.2% and 49.1% at 100 µM, respectively. Docking study manifested that 6 well located in the catalytic pocket of TGR5 by hydrogen-bond and hydrophobic interactions. The GLP-1 promotion of 6 could be attenuated by IP3, Ca2+/CaMKII and MEK/ERK pathway inhibitors, suggesting that these pathways played important roles in GLP-1 secretion. Thus, stilbenes in peony seeds maybe regarded as potential GLP-1 secretagogues through TGR5-IP3-Ca2+/CaMKII-MEK/ERK pathways.

193-nm photodissociation of singly and multiply charged peptide anions for acidic proteome characterization.

193-nm ultraviolet photodissociation (UVPD) was implemented to sequence singly and multiply charged peptide anions. Upon dissociation by this method, a-/x-type, followed by d and w side-chain loss ions, were the most prolific and abundant sequence ions, often yielding 100% sequence coverage. The dissociation behavior of singly and multiply charged anions was significantly different with higher charged precursors yielding more sequence ions; however, all charge states investigated (1- through 3-) produced rich diagnostic information. UVPD at 193 nm was also shown to successfully differentiate and pinpoint labile phosphorylation modifications. The sequence ions were produced with high abundances, requiring limited averaging for satisfactory spectral quality. The intact, charge-reduced radical products generated by UV photoexcitation were also subjected to collision-induced dissociation (termed, activated-electron photodetachment dissociation (a-EPD)), but UVPD alone yielded more predictable and higher abundance sequence ions. With the use of a basic (pH∼11.5), piperidine-modified mobile phase, LC-MS/UVPD was implemented and resulted in the successful analysis of mitogen-activated pathway kinases (MAPKs) using ultrafast activation times (5 ns).

The role of mechanical stretching in the activation and localization of adhesion proteins and related intracellular molecules.

The molecular complexity of the processes which lead to cell adhesion includes membrane and cytoskeletal proteins, involved in the focal adhesion formation, as well as signaling molecules tightly associated with the main intracellular regulatory cascades (Akt/PKB and MAPK/Erk). Dynamic environments, which create substrate deformations at determined frequencies and timing, have significant influences on adhesion mechanisms and in general in cellular behavior. In this work, we investigated the role of mechanical stretching (10% substrate deformation, 1 Hz frequency applied up to 60 min) on adhesion proteins (vinculin and focal adhesion kinase-FAK), related RhoGTPases (Rac1 and RhoA), and intracellular pathways (Akt/PKB and MAPK/Erk) in terms of activation and membrane recruitment in relation with cytoskeletal changes observed (membrane ruffling and filopodia formation). These changes are due to intracellular molecular rearrangements, acting with sequential concerted dynamics, able to modify the cytoskeletal conformation. The observed cellular response adds some important issues for better understanding the cellular behavior in environment which mimic as close as possible the physiological conditions.

PPAR-gamma agonist ameliorates kidney and liver disease in an orthologous rat model of human autosomal recessive polycystic kidney disease.

In autosomal recessive polycystic kidney disease (ARPKD), progressive enlargement of fluid-filled cysts is due to aberrant proliferation of tubule epithelial cells and transepithelial fluid secretion leading to extensive nephron loss and interstitial fibrosis. Congenital hepatic fibrosis associated with biliary cysts/dilatations is the most common extrarenal manifestation in ARPKD and can lead to massive liver enlargement. Peroxisome proliferator-activated receptor γ (PPAR-γ), a member of the ligand-dependent nuclear receptor superfamily, is expressed in a variety of tissues, including the kidneys and liver, and plays important roles in cell proliferation, fibrosis, and inflammation. In the current study, we determined that pioglitazone (PIO), a PPAR-γ agonist, decreases polycystic kidney and liver disease progression in the polycystic kidney rat, an orthologous model of human ARPKD. Daily treatment with 10 mg/kg PIO for 16 wk decreased kidney weight (% of body weight), renal cystic area, serum urea nitrogen, and the number of Ki67-, pERK1/2-, and pS6-positive cells in the kidney. There was also a decrease in liver weight (% of body weight), liver cystic area, fibrotic index, and the number of Ki67-, pERK1/2-, pERK5-, and TGF-ß-positive cells in the liver. Taken together, these data suggest that PIO inhibits the progression of polycystic kidney and liver disease in a model of human ARPKD by inhibiting cell proliferation and fibrosis. These findings suggest that PPAR-γ agonists may have therapeutic value in the treatment of the renal and hepatic manifestations of ARPKD.

Early detection of neuropathophysiology using diffusion-weighted magnetic resonance imaging in asymptomatic cats with feline immunodeficiency viral infection.

HIV infection results in a highly prevalent syndrome of cognitive and motor disorders designated as HIV-associated dementia (HAD). Neurologic dysfunction resembling HAD has been documented in cats infected with strain PPR of the feline immunodeficiency virus (FIV), whereas another highly pathogenic strain (C36) has not been known to cause neurologic signs. Animals experimentally infected with equivalent doses of FIV-C36 or FIV-PPR, and uninfected controls were evaluated by magnetic resonance diffusion-weighted imaging (DW-MRI) and spectroscopy (MRS) at 17.5-18 weeks post-infection, as part of a study of viral clade pathogenesis in FIV-infected cats. The goals of the MR imaging portion of the project were to determine whether this methodology was capable of detecting early neuropathophysiology in the absence of outward manifestation of neurological signs and to compare the MR imaging results for the two viral strains expected to have differing degrees of neurologic effects. We hypothesized that there would be increased diffusion, evidenced by the apparent diffusion coefficient as measured by DW-MRI, and altered metabolite ratios measured by MRS, in the brains of FIV-PPR-infected cats relative to C36-infected cats and uninfected controls. Increased apparent diffusion coefficients were seen in the white matter, gray matter, and basal ganglia of both the PPR and C36-infected (asymptomatic) cats. Thalamic MRS metabolite ratios did not differ between groups. The equivalently increased diffusion by DW-MRI suggests similar indirect neurotoxicity mechanisms for the two viral genotypes. DW-MRI is a sensitive tool to detect neuropathophysiological changes in vivo that could be useful during longitudinal studies of FIV.

Polarized localization of yeast Pbs2 depends on osmostress, the membrane protein Sho1 and Cdc42.

In Saccharomyces cerevisiae cells, high external osmolarity leads to the activation of a p38-related mitogen-activated protein (MAP) kinase though Pbs2. Pbs2 tagged with green fluorescent protein (Pbs2-GFP) is evenly distributed in the cytoplasm but excluded from the nucleus before and after exposure to stress. Here we show that a catalytically inactive form of Pbs2 attains a highly polarised localization during osmostress. This phenomenon depends of the osmosensor Sho1 and on a functional Cdc42 GTPase. Cdc42, but not the actin cytoskeleton, influences Sho1-dependent activation of the MAP kinase. Sho1 itself accumulates at sites of polar growth, but independently of stress conditions and Cdc42. These observations allow us to define the sequence of events that occurs during propogation of osmostress signals.

Signal transduction and metabolism in chondrocytes is modulated by lactoferrin.

OBJECTIVE: Activation of granulocytes causes a considerable rise in the concentration of lactoferrin (Lf) in synovial fluid (SF). We here investigate consequences thereof on signal transduction and the balance between catabolic and anabolic metabolism in chondrocytes. METHODS: Signal transduction was analysed in cultured chondrocytes by immunodetection of mitogen activated protein kinases (MAPK) and analysis of Smad2 translocation to the nucleus. Expression levels of matrix metalloproteinases (MMPs) and of aggrecan were measured by reverse-transcription-PCR. The proteolytic activity of MMPs was ascertained by zymography. Expression of the low-density-lipoprotein-receptor-related-protein-1 (LRP-1), a Lf receptor for signalling, was assayed by immunohistochemistry in cartilage and in cultured chondrocytes by immunoblotting. RESULTS: We found LRP-1 expressed in dedifferentiated chondrocytes in culture and in cartilage tissue preferentially on the articular surface where it can encounter Lf within SF. Lf stimulated proliferation of chondrocytes, comparable to transforming growth factor-beta1 (TGFbeta1) and activated p38 and the extracellular-signal regulated-kinases 1/2 (ERK1/2) within minutes. Surprisingly, Lf induced nuclear Smad2 translocation, a signal pathway ascribed to TGFbeta receptor activation. Lf significantly increased the levels of catabolic indicators such as MMP1, MMP2, MMP3 and MMP13 and inhibited aggrecan synthesis. CONCLUSION: Lf is a robust regulator of chondrocyte metabolism, comparable to TGFbeta1. The catabolic influence together with the proliferative stimulus indicates a function as an early phase cytokine, enhancing MMPs, necessary for degradation of damaged tissue and stimulating proliferation of chondrocytes, necessary for reconstruction.

Hepatic regenerative response in small-sized liver isografts in the rat.

BACKGROUND: To investigate hepatic regenerative response and associated mechanisms in different-size liver grafts in the rat. METHODS: Rat models of different-size-graft liver transplantation (whole, 50%-size, or 30%-size) were established, with a sham operation group serving as a control. Portal pressure, graft injury, interleukin 6 (IL-6), signal transducer and activator of transcription (Stat3), mitogen-activated protein kinase (MAPK), cyclin D1, and proliferating cell nuclear antigen (PCNA) were all assessed. RESULTS: The portal pressure was significantly higher and hepatic injury more severe in the smaller sized groups than in the whole graft group, especially in the 30%-size grafts. Hepatic IL-6 and tumor necrosis factor-alpha (TNF-alpha) levels in the two smaller sized groups were significantly higher than in the whole graft group, while IL-6 levels appeared to be negatively associated with graft sizes. Downstream markers of IL-6, Stat3 and MAPK phosphorylation, cyclin D1, and PCNA expression were also markedly increased in the small-sized grafts compared with the whole grafts, and appeared to positively correlate with early measurements of portal pressure and subsequent hepatic injury. CONCLUSION: Vigorous hepatic regeneration in small-for-size liver grafts may be associated with highly activated IL-6/Stat3 and MAPK signaling, which may in turn correlate with graft size, portal pressure, and hepatic injury.

Vaccarin hastens wound healing by promoting angiogenesis via activation of MAPK/ERK and PI3K/AKT signaling pathways in vivo.

PURPOSE: To explore the potential role and unclear molecular mechanisms of vaccarin in wound healing. METHODS: Rats' skin excision model to study the effects of vaccarin on wound healing in vivo . Hematoxylin and eosin staining was performed to evaluate Histopathologic characteristics. Immunohistochemistry was employed to assess the effects of vaccarin in accelerating angiogenesis. Western blot was used to evaluate relative protein expressed levels. RESULTS: Vaccarin could significantly promote wound healing and endothelial cells and fibroblasts proliferation in the wound site. Immunohistochemistry and Western blot studies showed that the nodal proteins and receptor (bFGFR) related to angiogenesis signaling pathway were activated, and the microvascular density in the wound site was markedly higher than that in the control group. CONCLUSIONS: The present study was the first to demonstrate that vaccarin is able to induce angiogenesis and accelerate wound healing in vivo by increasing expressions of p-Akt, p-Erk and p-bFGFR. This process is mediated by MAPK/ERK and PI3K/AKT signaling pathways.

Protein expression pattern of murine macrophages treated with anthrax lethal toxin.

Anthrax lethal toxin (LeTx; a combination of protective antigen and lethal factor) secreted by the vegetative cells of Bacillus anthracis is cytotoxic for certain macrophage cell lines. The role of LeTx in mediating these effects is complicated largely due to the difficulty in identifying and assigning functions to the affected proteins. To analyze the protein profile of murine macrophages treated with LeTx, we employed two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF MS, and interpreted the peptide mass fingerprint data relying on the ProFound database. Among the differentially expressed spots, cleaved mitogen-activated protein kinase kinase 1 acting as a negative element in the signal transduction pathway, and glucose-6-phosphate dehydrogenase playing a role in the protection of cells from hyperproduction of active oxygen were up-regulated in the LeTx-treated macrophages.

Glycogen synthase kinase 3B in bovine oocytes and granulosa cells: possible involvement in meiosis during in vitro maturation.

Glycogen synthase kinase 3 (GSK3) regulates cellular metabolism and cell cycle via different signalling pathways. In response to insulin and growth factors GSK3 is serine-phosphorylated and inactivated. We analysed GSK3B expression and activation in bovine cumulus cells (CC) and oocytes at different meiotic stages in vitro in parallel with MAP kinases ERK (MAPK3/MAPK1) and p38 (MAPK14). GSK3B localised to cytoplasm in granulosa cells and in oocytes throughout folliculogenesis. In mature metaphase-II (MII) oocytes, GSK3B was concentrated to the region of midzone between the oocyte and the first polar body, as well as active phospho-Thr Aurora A kinase (AURKA). During in vitro maturation (IVM), in oocytes, phospho-Ser(9)-GSK3B level increased as well as phospho-MAPK3/MAPK1, while phospho-MAPK14 decreased. In CC, phospho-MAPK14 increased upon germinal vesicle breakdown (GVBD)/metaphase-I (MI) and then decreased during transition to MII. Administration of inhibitors of GSK3 activity (lithium chloride or 2'Z,3'E -6-bromoindirubin-3'-oxime) rapidly increased phospho-Ser(9)-GSK3B, and led to transient decrease of phospho-MAPK3/MAPK1 and to durable enhancing of phospho-MAPK14 in granulosa primary cell culture. GSK3 inhibitors during IVM diminished cumulus expansion and delayed meiotic progression. In cumulus, phospho-MAPK14 level was significantly higher in the presence of inhibitors, comparing with control, through the time of MI/MII transition. In oocytes, phospho-GSK3B was increased and phospho-MAPK3/MAPK1 was decreased before GVBD and oocytes were mainly arrested at MI. Therefore, GSK3B might regulate oocyte meiosis, notably MI/MII transition being the part of MAPK3/1 and MAPK14 pathways in oocytes and CC. GSK3B might be also involved in the local activation of AURKA that controls this transition.

Spa-RQ: an Image Analysis Tool to Visualise and Quantify Spatial Phenotypes Applied to Non-Small Cell Lung Cancer.

To facilitate analysis of spatial tissue phenotypes, we created an open-source tool package named 'Spa-RQ' for 'Spatial tissue analysis: image Registration & Quantification'. Spa-RQ contains software for image registration (Spa-R) and quantitative analysis of DAB staining overlap (Spa-Q). It provides an easy-to-implement workflow for serial sectioning and staining as an alternative to multiplexed techniques. To demonstrate Spa-RQ's applicability, we analysed the spatial aspects of oncogenic KRAS-related signalling activities in non-small cell lung cancer (NSCLC). Using Spa-R in conjunction with ImageJ/Fiji, we first performed annotation-guided tumour-by-tumour phenotyping using multiple signalling markers. This analysis showed histopathology-selective activation of PI3K/AKT and MAPK signalling in Kras mutant murine tumours, as well as high p38MAPK stress signalling in p53 null murine NSCLC. Subsequently, Spa-RQ was applied to measure the co-activation of MAPK, AKT, and their mutual effector mTOR pathway in individual tumours. Both murine and clinical NSCLC samples could be stratified into 'MAPK/mTOR', 'AKT/mTOR', and 'Null' signature subclasses, suggesting mutually exclusive MAPK and AKT signalling activities. Spa-RQ thus provides a robust and easy to use tool that can be employed to identify spatially-distributed tissue phenotypes.