Interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to activate JAK2 in various cells, but the role of JAK2 in IL-3 or GM-CSF receptor signal transduction is largely unknown. We have now examined the role of JAK2 in GM-CSF-induced signaling events in BA/F3 cells. In BA/F3 cells expressing hGMR, activation of JAK2 by hGM-CSF requires the box1 region of hGMR beta. Dominant negative JAK2 (delta JAK2), which lacked the kinase domain suppressed mIL-3 or hGM-CSF-induced c-fos promoter activation as well as c-myc promoter activation/cell proliferation, thereby suggesting that JAK2 is involved in the signaling of both pathways. Further analyses of the role of JAK2 in c-fos gene activation in BA/F3 cells expressing hGMR revealed that delta JAK2 inhibited hGM-CSF-induced phosphorylation of Shc and protein tyrosine phosphatase 1D. Within hGMR beta, the several tyrosine residues which exist are related to activation of Shc or protein tyrosine phosphate 1D, and are phosphorylated in response to hGM-CSF stimulation. In addition, we observed that delta JAK2 inhibited hGM-CSF-induced phosphorylation of hGMR beta. Taken together, our results suggest that JAK2 activated by the box1 region of hGMR mediates hGM-CSF-induced c-fos promoter activation through phosphorylation of hGMR.

Formation of nodes of Ranvier requires contact of axons with myelinating glial cells, generating specialized axo-glial subdomains. Caspr/paranodin is required for the formation of septate-like junctions at paranodes, whereas the related caspr2 is essential for the organization of juxtaparanodes. The molecular mechanisms underlying the segregation of these related glycoproteins within distinct complexes are poorly understood. Exit of paranodin from the endoplasmic reticulum (ER) is mediated by its interaction with F3/contactin. Using domain swapping with caspr2, we mapped a motif with Pro-Gly-Tyr repeats (PGY) in the ectodomain of paranodin responsible for its ER retention. Deletion of PGY allows cell surface delivery of paranodin bypassing the calnexin-calreticulin quality control. Conversely, insertion of PGY in caspr2 or NrCAM blocks these proteins in the ER. PGY is a novel type of processing signal that compels chaperoning of paranodin by contactin. Contactin associated with paranodin is expressed at the cell surface with high-mannose N-glycans. Using mutant CHO lines altered in the processing of N-linked carbohydrates, we show that the high-mannose glycoform of contactin strongly binds neurofascin-155, its glial partner at paranodes. Thus, the unconventional processing of paranodin and contactin may determine the selective association of axo-glial complexes at paranodes.

OBJECTIVE

This study aimed to enhance the diagnostic accuracy by defining different cutoff liver stiffness measurement (LSM) values according to alanine aminotransferase (ALT) level and combining LSM with noninvasive models in patients with chronic hepatitis B (CHB).

BACKGROUND

Several studies have indicated that ALT influences LSM using FibroScan.

METHODS

The study prospectively enrolled 200 patients (143 men, mean age 45.4 y) between June 2007 and November 2008 who had been diagnosed with CHB and underwent both liver biopsy and LSM on the same day.

RESULTS

The area under the receiver operating characteristic curves (AUROC) of LSM for predicting cirrhosis in patients with ALT < or = upper limit of normal (ULN) was higher than that of all patients or those with ALT>>ULN and < or = 2x ULN (AUROC=0.884 vs. 0.849 and 0.867). The cutoff LSM values for>> or = F2,>> or = F3, and F4 were 6.0, 7.5, and 10.1 kPa, respectively, in patients with ALT < or = ULN, whereas they were 8.9, 11.0, and 15.5 kPa, respectively, in those with ALT>>ULN and < or = 2x ULN. The combination of LSM and the age-spleen-platelet ratio index performed the best at predicting cirrhosis, regardless of ALT level (AUROC=0.917 in patients with ALT < or = ULN, 0.909 in those with ALT < or = 2x ULN, and 0.894 in all patients).

CONCLUSIONS

Different cutoff LSM values according to ALT level and combination with age-spleen-platelet ratio index can enhance the performance of LSM in CHB, regardless of ALT level.

Background: Aging has often been linked to age-related vascular disorders. The elucidation of the putative genes and pathways underlying vascular aging likely provides useful insights into vascular diseases at advanced ages. Transcriptional regulatory network analysis is the key to describing genetic interactions between molecular regulators and their target gene transcriptionally changed during vascular aging.Results: A total of 469 differentially expressed genes were parsed into 6 modules. Among the incorporated sample traits, the most significant module related to vascular aging was associated with triglyceride and enriched with biological terms like proteolysis, blood circulation, and circulatory system process. The module associated with triglyceride was preserved in an independent microarray dataset, indicating the robustness of the identified vascular aging-related subnetwork. Additionally, Enpp5, Fez1, Kif1a, F3, H2-Q7, and their interacting miRNAs mmu-miR-449a, mmu-miR-449c, mmu-miR-34c, mmu-miR-34b-5p, mmu-miR-15a, and mmu-let-7, exhibited the most connectivity with external lipid-related traits. Transcriptional alterations of the hub genes Enpp5, Fez1, Kif1a, and F3, and the interacting microRNAs mmu-miR-34c, mmu-miR-34b-5p, mmu-let-7, mmu-miR-449a, and mmu-miR-449c were confirmed.Conclusion: Our findings demonstrate that triglyceride and free fatty acid-related genes are key regulators of age-related vascular dysfunction in mice and show that the hub genes for Enpp5, Fez1, Kif1a, and F3 as well as their interacting miRNAs mmu-miR-34c, mmu-miR-34b-5p, mmu-let-7, mmu-miR-449a, and mmu-miR-449c, could serve as potential biomarkers in vascular aging.Methods: The microarray gene expression profiles of aorta samples from 6-month old mice (n=6) and 20-month old mice (n=6) were processed to identify nominal differentially expressed genes. These nominal differentially expressed genes were subjected to a weighted gene co-expression network analysis. A network-driven integrative analysis with microRNAs and transcription factors was performed to define significant modules and underlying regulatory pathways associated with vascular aging, and module preservation test was conducted to validate the age-related modules based on an independent microarray gene expression dataset in mice aorta samples including three 32-week old wild-type mice (around 6-month old) and three 78-week old wild-type mice (around 20-month old). Gene ontology and protein-protein interaction analyses were conducted to determine the hub genes as potential biomarkers in the progress of vascular aging. The hub genes were further validated with quantitative real-time polymerase chain reaction in aorta samples from 20 young (6-month old) mice and 20 old (20-month old) mice.

The oligodendrocyte-derived extracellular matrix protein J1-160/180 displays repellent substrate properties toward neurons. In a search for neuronal ligands mediating the response to J1-160/180, we have identified the F3/11 cell surface protein, a glyco-phosphatidylinositol-anchored member of the immunoglobulin superfamily. F3/11 mediates the initial recognition between a J1-160/180 substrate and cerebellar neurons or F3-transfected CHO cells. In cerebellar neurons, the F3/11-J1-160/180 interaction induces a repulsion consisting of the loss of substrate adhesion with time in culture and inhibition of neurite outgrowth. Antibody blocking experiments show that the avoidance response of neurites at J1-160/180 substrate borders is also mediated by F3/11. Active cell-cell and cell-substrate repulsion is considered a major mechanism governing the extent and directionality of axonal growth, but the ligand-receptor interactions involved have remained unknown. Our results show that F3/11 mediates the neuronal response to the repellent molecule J1-160/180 and may thus be involved in signal transduction leading to cell repulsion.

The effects of consumption of low doses of ethanol (0.75 mg/kg) on spectral components of the EEG were investigated in 24 21-25-year-old males. A comparison of mean spectral power in 4 frequency bands (4-7 Hz, 7.5-9 Hz, 9-12 Hz, and 12-20 Hz) at 90 min post consumption revealed that ethanol, as opposed to placebo, significantly increased power in the two slower frequency bands, in both posterior and anterior leads. A reduction in the frequency at which power values peaked in the theta (4-7 Hz), fast alpha (9-12 Hz), and beta (12-20 Hz) frequency ranges was also seen following ethanol consumption. The ethanol induced lowering of peak power in the fast alpha range was observed in all 4 leads (F4-C4, P4-O2, F3-C3, and P3-O1), whereas the frequency changes in the theta and beta frequencies were only significant in a frontal lead. Ethanol was also noted to produce a decrease in the stability of the EEG in the fast alpha band as quantified by the coefficient of variation of power. Subjective response to ethanol was observed to vary depending on the subjects' pre-drug EEG pattern. Men with high amounts of fast alpha activity at baseline, in lead P4-O2, reported feeling less 'high' and less overall feelings of intoxication after ethanol than the men with lower amounts of pre-drug fast alpha waves. In addition, subjects were sorted into 'low' and 'moderate' drinkers depending on their quantity and frequency of drinking over the previous 6 month period.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

To compare the diagnostic accuracy of TE and MRE and establish cutoff levels and diagnostic strategies for both techniques, enabling selection of patients for liver biopsy.

METHODS

One hundred three patients with chronic hepatitis B or C and liver biopsy were prospectively included. Areas under curves (AUROC) were compared for TE and MRE for METAVIR fibrosis grade ≥ F2 and ≥F3. We defined cutoff values for selection of patients with F0-F1 (sensitivity >95%) and for significant fibrosis F2-F4 (specificity >95%).

RESULTS

Following exclusions, 85 patients were analysed (65 CHB, 19 CHC, 1 co-infected). Fibrosis stages were F0 (n = 3), F1 (n = 53), F2 (n = 15), F3 (n = 8) and F4 (n = 6). TE and MRE accuracy were comparable [AUROCTE ≥ F2: 0.914 (95% CI: 0.857-0.972) vs. AUROCMRE ≥ F2: 0.909 (0.840-0.977), P = 0.89; AUROCTE ≥ F3: 0.895 (0.816-0.974) vs. AUROCMRE ≥ F3: 0.928 (0.874-0.982), P = 0.42]. Cutoff values of <5.2 and ≥8.9 kPa (TE) and <1.66 and ≥2.18 kPa (MRE) diagnosed 64% and 66% of patients correctly as F0-F1 or F2-F4. A conditional strategy in inconclusive test results increased diagnostic yield to 80%.

CONCLUSIONS

TE and MRE have comparable accuracy for detecting significant fibrosis, which was reliably detected or excluded in two-thirds of patients. A conditional strategy further increased diagnostic yield to 80%.

CONCLUSIONS

• Both ultrasound-based transient elastography and magnetic resonance elastography can assess hepatic fibrosis. • Both have comparable accuracy for detecting liver fibrosis in viral hepatitis. • The individual techniques reliably detect or exclude significant liver fibrosis in 66 %. • A conditional strategy for inconclusive findings increases the number of correct diagnoses.

Ascochyta blight, caused by the fungus Ascochyta rabiei (Pass.) Lab., is one of the most devastating diseases of chickpea (Cicer arietinum L.) worldwide. Research was conducted to map genetic factors for resistance to ascochyta blight using a linkage map constructed with 144 simple sequence repeat markers and 1 morphological marker (fc, flower colour). Stem cutting was used to vegetatively propagate 186 F2 plants derived from a cross between Cicer arietinum L. 'ICCV96029' and 'CDC Frontier'. A total of 556 cutting-derived plants were evaluated for their reaction to ascochyta blight under controlled conditions. Disease reaction of the F1 and F2 plants demonstrated that the resistance was dominantly inherited. A Fain's test based on the means and variances of the ascochyta blight reaction of the F3 families showed that a few genes were segregating in the population. Composite interval mapping identified 3 genomic regions that were associated with the reaction to ascochyta blight. One quantitative trait locus (QTL) on each of LG3, LG4, and LG6 accounted for 13%, 29%, and 12%, respectively, of the total estimated phenotypic variation for the reaction to ascochyta blight. Together, these loci controlled 56% of the total estimated phenotypic variation. The QTL on LG4 and LG6 were in common with the previously reported QTL for ascochyta blight resistance, whereas the QTL on LG3 was unique to the current population.

A significant level of root elongation was induced in rice (Oryza sativa) grown under phosphorus-deficient conditions. The root elongation clearly varied among a total of 62 varieties screened under two different phosphorus levels. Two contrasting varieties, 'Gimbozu', with a low elongating response and 'Kasalath', with a high elongating response, were chosen and crossed to produce a hybrid population for QTL analyses. QTLs for the phosphorus deficiency-induced root elongation were detected by two linkage maps, i.e., one with 82 F3 families constructed by 97 simple sequence repeat (SSR) and sequence-tag site markers and another with 97 F8 lines by 790 amplified fragment length polymorphism and SSR markers. A single QTL for the elongation response was detected on chromosome 6, with a LOD score of 4.5 in both maps and explained about 20% of total phenotypic variance. In addition, this QTL itself, or a region tightly linked with it, partly explained an ability to reduce accumulation of excess iron in the shoots. The identified QTL will be useful to improve rice varieties against a complex nutritional disorder caused by phosphorus deficiency and iron toxicity.

CPT-11 (irinotecan) has been investigated as a treatment for malignant brain tumors. However, limitations of CPT-11 therapy include low levels of the drug entering brain tumor sites and systemic toxicities associated with higher doses. Neural stem cells (NSCs) offer a novel way to overcome these obstacles because of their inherent tumor tropism and ability to cross the blood-brain barrier, which enables them to selectively target brain tumor sites. Carboxylesterases (CEs) are enzymes that can convert the prodrug CPT-11 (irinotecan) to its active metabolite SN-38, a potent topoisomerase I inhibitor. We have adenovirally transduced an established clonal human NSC line (HB1.F3.CD) to express a rabbit carboxylesterase (rCE) or a modified human CE (hCE1m6), which are more effective at converting CPT-11 to SN-38 than endogenous human CE. We hypothesized that NSC-mediated CE/CPT-11 therapy would allow tumor-localized production of SN-38 and significantly increase the therapeutic efficacy of irinotecan. Here, we report that transduced NSCs transiently expressed high levels of active CE enzymes, retained their tumor-tropic properties, and mediated an increase in the cytotoxicity of CPT-11 toward glioma cells. CE-expressing NSCs (NSC.CEs), whether administered intracranially or intravenously, delivered CE to orthotopic human glioma xenografts in mice. NSC-delivered CE catalyzed conversion of CPT-11 to SN-38 locally at tumor sites. These studies demonstrate the feasibility of NSC-mediated delivery of CE to glioma and lay the foundation for translational studies of this therapeutic paradigm to improve clinical outcome and quality of life in patients with malignant brain tumors.

OBJECTIVE

To evaluate the accuracy of shear-wave elastography (SWE) for staging liver fibrosis in patients with diffuse liver disease (including patients with hepatitis C virus [HCV]) and to determine the relative accuracy of SWE measurements obtained from different hepatic acquisition sites for staging liver fibrosis.

METHODS

The institutional review board approved this single-institution prospective study, which was performed between January 2010 and March 2013 in 136 consecutive patients who underwent SWE before their scheduled liver biopsy (age range, 18-76 years; mean age, 49 years; 70 men, 66 women). Informed consent was obtained from all patients. SWE measurements were obtained at four sites in the liver. Biopsy specimens were reviewed in a blinded manner by a pathologist using METAVIR criteria. SWE measurements and biopsy results were compared by using the Spearman correlation and receiver operating characteristic (ROC) curve analysis.

RESULTS

SWE values obtained at the upper right lobe showed the highest correlation with estimation of fibrosis (r = 0.41, P < .001). Inflammation and steatosis did not show any correlation with SWE values except for values from the left lobe, which showed correlation with steatosis (r = 0.24, P = .004). The area under the ROC curve (AUC) in the differentiation of stage F2 fibrosis or greater, stage F3 fibrosis or greater, and stage F4 fibrosis was 0.77 (95% confidence interval [CI]: 0.68, 0.86), 0.82 (95% CI: 0.75, 0.91), and 0.82 (95% CI: 0.70, 0.95), respectively, for all subjects who underwent liver biopsy. The corresponding AUCs for the subset of patients with HCV were 0.80 (95% CI: 0.67, 0.92), 0.82 (95% CI: 0.70, 0.95), and 0.89 (95% CI: 0.73, 1.00). The adjusted AUCs for differentiating stage F2 or greater fibrosis in patients with chronic liver disease and those with HCV were 0.84 and 0.87, respectively.

CONCLUSIONS

SWE estimates of liver stiffness obtained from the right upper lobe showed the best correlation with liver fibrosis severity and can potentially be used as a noninvasive test to differentiate intermediate degrees of liver fibrosis in patients with liver disease.

BACKGROUND

The purpose of this study was to compare the flexibility and cyclic fatigue resistance of ProTaper Universal (PTU; Dentsply Tulsa Dental Specialities, Tulsa, OK) and ProTaper Gold (PTG; Dentsply Tulsa Dental Specialities, Tulsa, OK) instruments in relation to their phase transformation behavior.

METHODS

Sizes S1, S2, F1, F2, and F3 of PTU and PTG instruments were subjected to rotational bending at a curvature of 40° and a radius of 6 mm. The number of cycles to fracture (NCF) was recorded. The fracture surface of all fragments was examined with a scanning electron microscope. Flexibility was determined by 45° bending tests according to the ISO 3630-1 specification. Unused and fractured instruments were examined by differential scanning calorimetry.

RESULTS

PTG had a cyclic fatigue resistance superior to PTU in all sizes (P < .001). The NCF of the nickel-titanium files of sizes S1 and S2 was significantly higher than those of sizes F1 to F3 (P < .001). No significant difference in the NCF of PTU instruments was detected between F1 and F2. The fractured files of both PTU and PTG showed the typical fracture pattern of fatigue failure. The bending load values were significantly lower for PTG than for PTU (P < .05). The differential scanning calorimetry analyses showed that each segment of the PTG instruments had a higher austenite finish temperature (50.1°C ± 1.7°C) than the PTU instruments (21.2°C ± 1.9°C) (P < .001). PTG instruments had a 2-stage transformation behavior. There was no significant difference in the austenite finish between unused files and instruments subjected to the fatigue process.

CONCLUSIONS

PTG files were significantly more flexible and resistant to fatigue than PTU files. PTG exhibited different phase transformation behavior than PTU, which may be attributed to the special heat treatment history of PTG instruments. PTG may be more suited for preparing canals with a more abrupt curvature.

OBJECTIVE

The aim of the present study was to compare the diagnostic performance of transient elastography (FibroScan) with that of serum fibrosis markers and stages of hepatic fibrosis by biopsy in 68 patients with chronic hepatitis B virus (HBV) and in 161 patients with hepatitis C virus (HCV) infection.

RESULTS

The serum levels of hyaluronic acid (r = 0.601) and type IV collagen (r = 0.663) significantly positively associated with the FibroScan values (all P < 0.05). Classified by fibrosis stages, the median values of FibroScan were 3.5 kPa for F0, 6.4 kPa for F1, 9.5 kPa for F2, 11.4 kPa for F3, and 15.4 kPa forF4 in patients with chronic HBV infection, and were 6.3 kPa for F0, 6.7 kPa for F1, 9.1 kPa for F2, 13.7 kPa for F3, and 26.4 kPa for F4 in those with chronic HCV infection. The values were significantly correlated with fibrosis stage for both (HBV, r = 0.559, P = 0.0093, and HCV, r = 0.686, P < 0.0001).

CONCLUSIONS

These results suggest that FibroScan is an efficient and simple method for evaluating liver fibrosis in patients with chronic infection, both for HBV and HCV.

Human 8p11 stem cell leukemia/lymphoma syndrome usually presents as a myeloproliferative disorder (MPD) that evolves to acute myeloid leukemia and/or lymphoma. The syndrome associated with t(8;13)(p11;q12) results in expression of the ZNF198-FGFR1 fusion tyrosine kinase that plays a pathogenic role in hematopoietic transformation. We found that ZNF198-FGFR1 activated both the AKT and mitogen activated protein kinase (MAPK) prosurvival signaling pathways, resulting in elevated phosphorylation of the AKT target FOXO3a at T32 and BAD at S112, respectively. These phosphorylated residues subsequently sequestered the proapoptotic FOXO3a and BAD to 14-3-3 to prevent apoptosis. We used a peptide-based 14-3-3 competitive antagonist, R18, to disrupt 14-3-3-ligand association. Expression of R18 effectively induced apoptosis in hematopoietic Ba/F3 cells transformed by ZNF198-FGFR1 compared with control cells. Moreover, purified recombinant transactivator of transcription (TAT)-conjugated R18 proteins effectively transduced into human leukemia cells and induced significant apoptosis in KG-1a cells expressing FGFR1OP2-FGFR1 fusion tyrosine kinase but not in control HL-60 and Jurkat T cells. Surprisingly, R18 was only able to dissociate FOXO3a, but not BAD as previously proposed, from 14-3-3 binding and induced apoptosis partially through liberation and reactivation of FOXO3a. Our findings suggest that 14-3-3 integrates prosurvival signals in FGFR1 fusion-transformed hematopoietic cells. Disrupting 14-3-3-ligand association may represent an effective therapeutic strategy to treat 8p11 stem cell MPD.

Epidemiological studies have reported associations between particulate matter (PM) concentrations and cancer and respiratory and cardiovascular diseases. DNA methylation has been identified as a possible link but so far it has only been analyzed in candidate sites.

We studied the association between DNA methylation and short- and mid-term air pollution exposure using genome-wide data and identified potential biological pathways for additional investigation.

We collected whole blood samples from three independent studies-KORA F3 (2004-2005) and F4 (2006-2008) in Germany, and the Normative Aging Study (1999-2007) in the United States-and measured genome-wide DNA methylation proportions with the Illumina 450k BeadChip. PM concentration was measured daily at fixed monitoring stations and three different trailing averages were considered and regressed against DNA methylation: 2-day, 7-day and 28-day. Meta-analysis was performed to pool the study-specific results.

Random-effect meta-analysis revealed 12 CpG (cytosine-guanine dinucleotide) sites as associated with PM concentration (1 for 2-day average, 1 for 7-day, and 10 for 28-day) at a genome-wide Bonferroni significance level (p ≤ 7.5E-8); 9 out of these 12 sites expressed increased methylation. Through estimation of I2 for homogeneity assessment across the studies, 4 of these sites (annotated in NSMAF, C1orf212, MSGN1, NXN) showed p>> 0.05 and I2 < 0.5: the site from the 7-day average results and 3 for the 28-day average. Applying false discovery rate, p-value < 0.05 was observed in 8 and 1,819 additional CpGs at 7- and 28-day average PM2.5 exposure respectively.

The PM-related CpG sites found in our study suggest novel plausible systemic pathways linking ambient PM exposure to adverse health effect through variations in DNA methylation.

Panni T, Mehta AJ, Schwartz JD, Baccarelli AA, Just AC, Wolf K, Wahl S, Cyrys J, Kunze S, Strauch K, Waldenberger M, Peters A. 2016. A genome-wide analysis of DNA methylation and fine particulate matter air pollution in three study populations: KORA F3, KORA F4, and the Normative Aging Study. Environ Health Perspect 124:983-990; http://dx.doi.org/10.1289/ehp.1509966.

In cucumber, Cucumis sativus L., the spine and skin colors are two important fruit quality traits for variety improvement. In this study, we investigated the inheritance of spine and mature fruit skin colors in F2 and F3 populations derived from a cross between two inbred lines WI7200 (black spine and orange fruit skin colors) and WI7201 (white spine and creamy fruit skin colors). We confirmed that a single, dominant gene, B, controlled both black spine color and orange mature fruit color. Initial framework mapping with microsatellite markers located the B locus in the distal region of the short arm of cucumber chromosome 4. Fine mapping was conducted with draft genome scaffold-assisted chromosome walking and stepwise increase of mapping population sizes, which allowed for the assignment of the B locus to a 50 kb genomic DNA region with two flanking markers that were 0.06 and 0.09 cM, respectively, from the B locus in a mapping population of 2,001 F2 plants. Gene annotation of this 50 kb region identified six genes including one encoding for a R2R3-MYB transcription factor. Sequence alignment of the R2R3-MYB homologs between the two parent inbreds identified a 1 bp deletion in the third intron of this gene in WI 7201. A molecular marker based on this indel was co-segregating with the spine and fruit colors. Quantitative RT-PCR revealed higher level of expression of this R2R3-MYB gene in WI7200 than in WI7201 in both immature and mature fruits. This R2R3-MYB gene seems to be the best candidate gene for the B locus conditioning black spine and orange mature fruit colors of cultivated cucumber.

IL-2R activates two distinct signaling pathways mediated by the adaptor protein Shc and the transcription factor STAT5. Prior mutagenesis studies of the IL-2R have indicated that the Shc and STAT5 pathways are redundant in the ability to induce lymphocyte proliferation. Yet paradoxically, T cells from STAT5-deficient mice fail to proliferate in response to IL-2, suggesting that the Shc pathway is unable to promote mitogenesis in the genetic absence of STAT5. Here we show in the murine lymphocyte cell line Ba/F3 that low levels of STAT5 activity are essential for Shc signaling. In the absence of STAT5 activity, Shc was unable to sustain activation of the Akt/p70S6 kinase pathway or promote lymphocyte proliferation and viability. Restoring STAT5 activity via a heterologous receptor rescued Shc-induced Akt/p70S6 kinase activity and cell proliferation with kinetics consistent with a transcriptional mechanism. Thus, STAT5 appears to regulate the expression of one or more unidentified components of the Akt pathway. Our results not only explain the severe proliferative defect in STAT5-deficient T cells but also provide mechanistic insight into the oncogenic properties of STAT5 in various leukemias and lymphomas.

Two components of the ERPs elicited by emotional visual stimuli, N300 and P300, were investigated. The emotional charge is explained through two dimensions: arousal (relaxing (R) or activating (A)) and valence (attractive (+) or repulsive (-)). Stimuli were slides of nudes (A+), human remains (A-), landscapes (R), and buildings (neutral (N)). The peculiar structure of the stimuli, along with a distracting task which allowed us to disguise the real aim of the experiment, helped to avoid a sort of 'relevance-for-task effect', mainly related to cognitive processes, which could explain P300 reactions in response to emotional visual stimuli found in several experiments. The ERPs were recorded from 32 subjects at F3, Fz, F4, C3, Cz, C4, P3, Pz and P4. In contrast to previous studies, P300 did not show greater amplitudes in response to emotional stimuli than to N. N300 showed greater amplitudes in response to A+ at parietal sites, the greatest differences being those with respect to A-. No inter-hemispheric differences were found. N300 confirms its usefulness as a variable for studying emotional reactions to visual stimuli.

Tea (Camellia sinensis (L.) O. Kuntze) leaves are a major source of flavonoids that mainly belong to the flavan-3-ols or catechins and are implicated in a wide range of health benefits. Although the catechins in tea leaves were identified long ago, the regulatory mechanisms governing catechin biosynthesis remain unclear. In the present work, the dynamic changes of catechin levels and the expression profiles of catechin-related genes in albino tea plants were intensively examined. The amounts of most catechins decreased to their lowest levels in the albino phase, when epigallocatechingallate was the highest of the catechins compared to all catechins, and catechin the lowest. Enzyme assays indicated that phenylalanine ammonia-lyase (PAL) activity was positively correlated with the concentration of catechins (r = 0.673). Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that the transcript abundance of flavonoid biosynthetic genes followed a tightly regulated biphasic pattern, and was affected by albinism. These genes (PAL, C4H, 4CL, CHS, CHI, F3H, FLS, F3'H, F3'5'H, DFR, LAR, ANS and ANR) encode enzymes in flavonoid biosynthesis. The expression levels of PAL, F3H and FLS were correlated with the concentration of catechins and the correlation coefficients were -0.683, 0.687 and -0.602, respectively. Therefore, these results indicate that PAL might be a core regulator in the control of catechin biosynthesis in albino tea plants.

Platelet integrin alphaIIbbeta3 activation is tightly controlled by intracellular signaling pathways, and several molecules, including talin, have been identified as critical for alphaIIbbeta3 activation. However, the whole pathway associated with alphaIIbbeta3 activation remains to be determined. To address this issue, we established a Chinese hamster ovary cell line (parental cells) that expresses constitutively activated chimeric integrin alphaIIbalpha6Bbeta3, and then obtained mutant cells expressing inactivated alphaIIbalpha6Bbeta3 by genome-wide mutagenesis. We have performed expression cloning to isolate signaling molecules responsible for integrin activation in the mutant cells. We show that integrin-linked kinase (ILK) complements defective integrin activation in the mutant cells. ILK mRNAs in the mutant cells contained 2 nonsense mutations, R317X and W383X, in a compound heterozygous state, resulting in a complete loss of ILK expression. Moreover, the mutant cells showed partially impaired activation of endogenous beta1 integrins. Knockdown of ILK in parental cells significantly suppressed the activated state of alphaIIbalpha6Bbeta3. However, ILK overexpression did not rescue the impaired integrin activation in talin knocked-down parental cells, whereas overexpression of talin-F3, a subdomain of the talin head domain, restored the function. Our present data suggest that ILK contributes to inside-out integrin activation.

CSF-1 regulates macrophage differentiation, survival, and function, and is an attractive therapeutic target for chronic inflammation and malignant diseases. Here we describe the effects of a potent and selective inhibitor of CSF-1R-CYC10268-on CSF-1R-dependent signaling. In in vitro kinase assays, CYC10268 was active in the low nanomolar range and showed selectivity over other kinases such as Abl and Kit. CYC10268 blocked survival mediated by CSF-1R in primary murine bone marrow-derived macrophages (BMM) and in the factor-dependent cell line Ba/F3, in which the CSF-1R was ectopically expressed. CYC10268 also inhibited CSF-1 regulated signaling (Akt, ERK-1/2), gene expression (urokinase plasminogen activator, toll-like receptor 9, and apolipoprotein E), and priming of LPS-inducible cytokine production in BMM. In thioglycollate-elicited peritoneal macrophages (TEPM), which survive in the absence of exogenous CSF-1, CYC10268 impaired LPS-induced cytokine production and regulated expression of known CSF-1 target genes. These observations support the conclusion that TEPM are CSF-1 autocrine and that CSF-1 plays a central role in macrophage effector functions during inflammation. CSF-1R inhibitors such as CYC10268 provide a powerful tool to dissect the role of the CSF-1/CSF-1R signaling system in a range of biological systems and have potential for a number of therapeutic applications.

There is currently no known genetic disease linked to prolactin (Prl) or its receptor (PrlR) in humans. Given the essential role of this hormonal system in breast physiology, we reasoned that genetic anomalies of Prl/PrlR genes may be related to the occurrence of breast diseases with high proliferative potential. Multiple fibroadenomas (MFA) are benign breast tumors which appear most frequently in young women, including at puberty, when Prl has well-recognized proliferative actions on the breast. In a prospective study involving 74 MFA patients and 170 control subjects, we identified four patients harboring a heterozygous single nucleotide polymorphism in exon 6 of the PrlR gene, encoding Ile(146)->>Leu substitution in its extracellular domain. This sole substitution was sufficient to confer constitutive activity to the receptor variant (PrlR(I146L)), as assessed in three reconstituted cell models (Ba/F3, HEK293 and MCF-7 cells) by Prl-independent (i) PrlR tyrosine phosphorylation, (ii) activation of signal transducer and activator of transcription 5 (STAT5) signaling, (iii) transcriptional activity toward a Prl-responsive reporter gene, and (iv) cell proliferation and protection from cell death. Constitutive activity of PrlR(I146L) in the breast sample from a patient was supported by increased STAT5 signaling. This is a unique description of a functional mutation of the PrlR associated with a human disease. Hallmarks of constitutive activity were all reversed by a specific PrlR antagonist, which opens potential therapeutic approaches for MFA, or any other disease that could be associated with this mutation in future.

ETV6/CBFA2 (TEL/AML1) is the most frequent genetic abnormality associated with acute lymphoblastic leukemias in children, and is associated with a favorable prognosis. To investigate the influence of ETV6/CBFA2 on cellular transformation, the fusion gene was cloned into a murine ecotropic retroviral vector and transduced into IL-3-dependent Ba/F3 and 32Dcl.3 and IL-7-dependent IxN/2b murine hematopoietic cell lines. Different variants of ETV6/CBFA2, corresponding to CBFA2 alternatively spliced variants, and the reciprocal product CBFA2/ETV6, were stably expressed in each of these cell lines. However, although Western blot analysis demonstrated expression of each variant, none of the stable cell lines expressing CBFA2/ETV6 or the variants conferred factor-independent growth. We further investigated the effect of ETV6/CBFA2 expression in vivo by generating transgenic mice in which expression of the fusion was directed to lymphoid cells using the immunoglobulin heavy chain enhancer/promoter. Four founder mice were identified showing transmission and expression of the chimeric product. The mice were bred for five generations and followed for more than 24 months. The mice did not develop a malignant hematologic disorder, nor did they display histopathologic, morphologic, or immunophenotypic abnormalities, although ETV6/CBFA2 expression was confirmed in each line. We conclude that the expression of ETV6/CBFA2 alone is not sufficient for induction of growth factor independence in hematopoietic cell lines or hematologic disease in transgenic mice.