Transforming growth factor-beta (TGF-beta) affects multiple cellular functions through the type I and type II receptor Ser/Thr kinases (TbetaRI and TbetaRII). Analysis of TGF-beta signaling pathways has been hampered by the lack of cell lines in which both TbetaRI and TbetaRII are deleted, and by the inability to study signal transduction by TbetaRI independently of TbetaRII since TbetaRI does not bind TGF-beta directly. To overcome these problems, we constructed and expressed chimeric receptors with the extracellular domain of the erythropoietin receptor (EpoR) and the cytoplasmic domains of TbetaRI or TbetaRII. When expressed in Ba/F3 cells, which do not express EpoR, Epo induces the formation of a heteromeric complex between cell surface EpoR-TbetaRI and EpoR-TbetaRII chimeras. Neither the EpoR-TbetaRI nor the EpoR-TbetaRII chimera interacts with endogenous TGF-beta receptors. Ba/F3 cells expressing both EpoR-TbetaRI and EpoR-TbetaRII chimeras, but not EpoR-TbetaRI or EpoR-TbetaRII alone, undergo Epo-induced growth arrest. When expressed in Ba/F3 cells in the absence of the EpoR-TbetaRII chimera, EpoR-TbetaRI(T204D), a chimeric receptor with a point mutation in the GS domain of TbetaRI that is autophosphorylated constitutively, triggers growth inhibition in response to Epo. Thus, both homo- and heterodimerization of the cytoplasmic domain of the type I TGF-beta receptor are required for intracellular signal transduction leading to inhibition of cell proliferation. These chimeric receptors provide a unique system to study the function and signal transduction of individual TGF-beta receptor subunits independently of endogenous TGF-beta receptors.

Liver fibrosis is currently assessed by liver biopsy, a costly and rather cumbersome procedure that is unsuitable for frequent patient monitoring, which drives research into biomarkers for this purpose. To investigate whether the serum N-glycome contains information suitable for this goal, we developed a 96-well plate-based serum N-glycomics sample preparation protocol that only involves fluid transfer steps and incubations in a PCR thermocycler yielding 8-aminopyrene-1,3,6-trisulfonic acid-labeled N-glycans. These N-glycans are then ready for analysis on the capillary electrophoresis-based DNA sequencers that are the current standard in clinical genetics laboratories worldwide. Subsequently we performed a multicenter, blinded study of 376 consecutive chronic hepatitis C virus patients for which liver biopsies and extensive serum biochemistry data were available. Among patients, the METAVIR fibrosis stage distribution was as follows: 10.6% F0, 44.4% F1, 20.5% F2, 18.4% F3, and 6.1% F4. We found that the ratio of two N-glycans, here called GlycoFibroTest, correlates with the histological fibrosis stage equally well as FibroTest (rho = 0.4-0.5 in F1-F4), which is used in the clinic today. Finally using affinity chromatography we depleted sera of immunoglobulin G, and this resulted in a complete removal of the undergalactosylated biantennary glycans from the N-glycome, which are partially determining GlycoFibroTest.

Rice is a model system for monocot but the molecular features of rice flavonoid biosynthesis have not been extensively characterized. Rice structural gene homologs encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) were identified by homology searches. Unique differential expression of OsF3H, OsDFR, and OsANS1 controlled by the Pl(w) locus, which contains the R/B-type regulatory genes OSB1 and OSB2, was demonstrated during light-induced anthocyanin accumulation in T65-Plw seedlings. Previously, F3H genes were often considered as early genes co-regulated with CHS and CHI genes in other plants. In selected non-pigmented rice lines, OSB2 is not expressed following illumination while their expressed OSB1sequences all contain the same nucleotide change leading to the T(64) M substitution within the conserved N-terminal interacting domain. Furthermore, the biochemical roles of the expressed rice structural genes (OsCHS1, OsCHI, OsF3H, and OsF3'H) were established in planta for the first time by complementation in the appropriate Arabidopsis transparent testa mutants. Using yeast two-hybrid analysis, OsCHS1 was demonstrated to interact physically with OsF3H, OsF3'H, OsDFR, and OsANS1, suggesting the existence of a macromolecular complex for anthocyanin biosynthesis in rice. Finally, flavones were identified as the major flavonoid class in the non-pigmented T65 seedlings in which the single-copy OsF3H gene was not expressed. Competition between flavone and anthocyanin pathways was evidenced by the significant reduction of tricin accumulation in the T65-Plw seedlings.

Interleukin-4 (IL-4) induces tyrosine phosphorylation of the latent transcription factor Stat6, which mediates the transcriptional responses of IL-4. The transactivation domain of Stat6 has recently been mapped to the C-terminal region of Stat6. We have investigated the mechanism by which Stat6, through its transactivation domain, induces transcription. Previous studies have shown that diverse regulated transcription factors interact with coactivators such as p300 and CBP. We report that Stat6 used the interaction with p300/CBP to exert its stimulatory effects. Overexpression of p300/CBP increased IL-4-induced transcription of Stat6 activated reporter genes. The requirement of p300/CBP for Stat6-mediated transactivation is shown by coexpression of the adenovirus E1A protein. E1A repressed the IL-4-induced reporter gene activity, while mutants of E1A, which do not interact with p300/CBP, failed to block the IL-4-induced response. In addition, we found that the minimal transactivation domain of Stat6, when fused to the GAL4 DNA-binding domain, was repressed by E1A, whereas the fusion protein p300-VP16 increased the transcriptional activity. In two-hybrid protein interaction assays in mammalian cells, we mapped the interaction domain of CBP to a C-terminal region between amino acids 1850 and 2176, a region distinct from the interaction domain of CBP with Stat1, Stat2 or Stat5. Finally, we show that antibodies raised against p300 coimmunoprecipitated Stat6 and p300 from transfected COS7 cells and antibodies against Stat6 coimmunprecipitated endogenous Stat6 and CBP from Ba/F3 cells. Our data suggest that the transactivation domain of Stat6 makes contact with the basal transcription machinery by binding to p300/CBP.

In the current study, we examined the types and frequency of KIT mutations in mast cell tumors from 191 dogs. Sequencing of reverse transcription-PCR products revealed alterations in 50 (26.2%) of the dogs. Most mutations were in exon 11 (n = 32), and of these, most were internal tandem duplications (n = 25) between residues 571 and 590. Within exon 11, there were two hotspots for mutations at codons 555-559 and 571-590. In addition, nine dogs had mutations in exon 8 and eight had mutations in exon 9. We selected the two most common mutants and two representative exon 11 mutants for further analysis. When expressed in Ba/F3 cells, they were constitutively tyrosine phosphorylated and induced growth factor-independent cell proliferation. AG1296, a tyrosine kinase inhibitor, dose dependently inhibited both the tyrosine phosphorylation of these mutants and their induction of growth factor-independent proliferation. This study shows that activating mutations in not only exon 11 but also exons 8 and 9 are common in canine mast cell tumors. These results also show that Ba/F3 cells can be used for the direct characterization of canine KIT mutants, eliminating the need to make equivalent mutations in the mouse or human genes.

Apoptotic cells are swiftly engulfed by macrophages to prevent the release of noxious materials from dying cells. Apoptotic cells expose phosphatidylserine (PtdSer) on their surface, and macrophages engulf them by recognizing PtdSer using specific receptors and opsonins. Here, we found that mouse resident peritoneal macrophages expressing Tim4 and MerTK are highly efficient at engulfing apoptotic cells. Neutralizing antibodies against either Tim4 or MerTK inhibited the macrophage engulfment of apoptotic cells. Tim4-null macrophages exhibited reduced binding and engulfment of apoptotic cells, whereas MerTK-null macrophages retained the ability to bind apoptotic cells but failed to engulf them. The incubation of wild-type peritoneal macrophages with apoptotic cells induced the rapid tyrosine phosphorylation of MerTK, which was not observed with Tim4-null macrophages. When mouse Ba/F3 cells were transformed with Tim4, apoptotic cells bound to the transformants but were not engulfed. Transformation of Ba/F3 cells with MerTK had no effect on the binding or engulfment of apoptotic cells; however, Tim4/MerTK transformants exhibited strong engulfment activity. Taken together, these results indicate that the engulfment of apoptotic cells by resident peritoneal macrophages proceeds in two steps: binding to Tim4, a PtdSer receptor, followed by MerTK-mediated cell engulfment.

BACKGROUND

The complex and dynamic changes during grape berry development have been studied in Vitis vinifera, but little is known about these processes in other Vitis species. The grape variety 'Norton', with a major portion of its genome derived from Vitis aestivalis, maintains high levels of malic acid and phenolic acids in the ripening berries in comparison with V. vinifera varieties such as Cabernet Sauvignon. Furthermore, Norton berries develop a remarkably high level of resistance to most fungal pathogens while Cabernet Sauvignon berries remain susceptible to those pathogens. The distinct characteristics of Norton and Cabernet Sauvignon merit a comprehensive analysis of transcriptional regulation and metabolite pathways.

RESULTS

A microarray study was conducted on transcriptome changes of Norton berry skin during the period of 37 to 127 days after bloom, which represents berry developmental phases from herbaceous growth to full ripeness. Samples of six berry developmental stages were collected. Analysis of the microarray data revealed that a total of 3,352 probe sets exhibited significant differences at transcript levels, with two-fold changes between at least two developmental stages. Expression profiles of defense-related genes showed a dynamic modulation of nucleotide-binding site-leucine-rich repeat (NBS-LRR) resistance genes and pathogenesis-related (PR) genes during berry development. Transcript levels of PR-1 in Norton berry skin clearly increased during the ripening phase. As in other grapevines, genes of the phenylpropanoid pathway were up-regulated in Norton as the berry developed. The most noticeable was the steady increase of transcript levels of stilbene synthase genes. Transcriptional patterns of six MYB transcription factors and eleven structural genes of the flavonoid pathway and profiles of anthocyanins and proanthocyanidins (PAs) during berry skin development were analyzed comparatively in Norton and Cabernet Sauvignon. Transcriptional patterns of MYB5A and MYB5B were similar during berry development between the two varieties, but those of MYBPA1 and MYBPA2 were strikingly different, demonstrating that the general flavonoid pathways are regulated under different MYB factors. The data showed that there were higher transcript levels of the genes encoding flavonoid-3'-O-hydroxylase (F3'H), flavonoid-3',5'-hydroxylase (F3'5'H), leucoanthocyanidin dioxygenase (LDOX), UDP-glucose:flavonoid 3'-O-glucosyltransferase (UFGT), anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR) 1 and LAR2 in berry skin of Norton than in those of Cabernet Sauvignon. It was also found that the total amount of anthocyanins was markedly higher in Norton than in Cabernet Sauvignon berry skin at harvest, and five anthocyanin derivatives and three PA compounds exhibited distinctive accumulation patterns in Norton berry skin.

CONCLUSIONS

This study provides an overview of the transcriptome changes and the flavonoid profiles in the berry skin of Norton, an important North American wine grape, during berry development. The steady increase of transcripts of PR-1 and stilbene synthase genes likely contributes to the developmentally regulated resistance during ripening of Norton berries. More studies are required to address the precise role of each stilbene synthase gene in berry development and disease resistance. Transcriptional regulation of MYBA1, MYBA2, MYB5A and MYBPA1 as well as expression levels of their putative targets F3'H, F3'5'H, LDOX, UFGT, ANR, LAR1, and LAR2 are highly correlated with the characteristic anthocyanin and PA profiles in Norton berry skin. These results reveal a unique pattern of the regulation of transcription and biosynthesis pathways underlying the viticultural and enological characteristics of Norton grape, and yield new insights into the understanding of the flavonoid pathway in non-vinifera grape varieties.

Ashwagandha, also called as "Queen of Ayurveda" and "Indian ginseng", is a commonly used plant in Indian traditional medicine, Ayurveda. Its roots have been used as herb remedy to treat a variety of ailments and to promote general wellness. However, scientific evidence to its effects is limited to only a small number of studies. We had previously identified anti-cancer activity in the leaf extract (i-Extract) of Ashwagandha and demonstrated withanone as a cancer inhibitory factor (i-Factor). In the present study, we fractionated the i-Extract to its components by silica gel column chromatography and subjected them to cell based activity analyses. We found that the cancer inhibitory leaf extract (i-Extract) has, at least, seven components that could cause cancer cell killing; i-Factor showed the highest selectivity for cancer cells and i-Factor rich Ashwagandha leaf powder was non-toxic and anti-tumorigenic in mice assays. We undertook a gene silencing and pathway analysis approach and found that i-Extract and its components kill cancer cells by at least five different pathways, viz. p53 signaling, GM-CFS signaling, death receptor signaling, apoptosis signaling and G2-M DNA damage regulation pathway. p53 signaling was most common. Visual analysis of p53 and mortalin staining pattern further revealed that i-Extract, fraction F1, fraction F4 and i-Factor caused an abrogation of mortalin-p53 interactions and reactivation of p53 function while the fractions F2, F3, F5 work through other mechanisms.

OBJECTIVE

To prospectively assess the utility of perfusion computed tomography (CT) for differentiating minimal from intermediate fibrosis in treatment-naïve patients with chronic hepatitis C virus (HCV) infection.

METHODS

This study was approved by the Institutional Review Board, and informed consent was obtained. Fifty-two patients with treatment-naïve HCV infection underwent perfusion CT and percutaneous liver biopsy on the same day. Portal vein, arterial, and total liver perfusion; mean transit time; and distribution volumes for the right and left liver lobes were measured. Liver samples were scored for fibrosis, and fibrosis area was determined. Differences in quantitative perfusion parameters between patients with minimal fibrosis (score of F1) and those with intermediate fibrosis (score of F2 or F3) were tested.

RESULTS

In patients with intermediate fibrosis (F2 and F3) compared with those with minimal fibrosis (F1), the portal venous perfusion (87 mL min(-1) 100 mL(-1) +/- 27 [standard deviation] vs 138 mL min(-1) 100 mL(-1) +/- 112, P = .042) and total liver perfusion (107 mL min(-1) 100 mL(-1) +/- 31 vs 169 mL min(-1) 100 mL(-1) +/- 137, P = .02) were significantly decreased, and the mean transit time was significantly increased (16 seconds +/- 4 vs 13 seconds +/- 5, P = .025). At multivariate analysis, only the mean transit time was an independent factor (odds ratio, 1.18; 95% confidence interval: 1.02, 1.37; P = .030). Receiver operating characteristic curve analysis showed that a mean transit time threshold of 13.4 seconds allowed discrimination between minimal and intermediate fibrosis with a sensitivity of 71% and a specificity of 65%.

CONCLUSIONS

The results of this study show that perfusion changes occur early during fibrosis in chronic HCV infection and can be detected with perfusion CT. Perfusion CT may help to discriminate minimal from intermediate fibrosis. Mean transit time appears to be the most promising perfusion parameter for differentiating between fibrosis stages, although the large amount of overlap in the measured parameters limits the clinical utility of this test at present.

OBJECTIVE

To compare the diagnostic accuracy of magnetic resonance imaging elastography (MRE) and anatomic MRI features in the diagnosis of severe hepatic fibrosis and cirrhosis.

METHODS

Three readers independently assessed presence of morphological changes associated with hepatic fibrosis in 72 patients with liver biopsy including: caudate to right lobe ratios, nodularity, portal venous hypertension (PVH) stigmata, posterior hepatic notch, expanded gallbladder fossa, and right hepatic vein caliber. Three readers measured shear stiffness values using quantitative shear stiffness maps (elastograms). Sensitivity, specificity, and diagnostic accuracy of stiffness values and each morphological feature were calculated. Interreader agreement was summarized using weighted kappa statistics. Intraclass correlation coefficient was used to assess interreader reproducibility of stiffness measurements. Binary logistic regression was used to assess interreader variability for dichotomized stiffness values and each morphological feature.

RESULTS

Using 5.9 kPa as a cutoff for differentiating F3-F4 from F0-2 stages, overall sensitivity, specificity, and diagnostic accuracy for MRE were 85.4%, 88.4%, and 87%, respectively. Overall interreader agreement for stiffness values was substantial, with an insignificant difference (P = 0.74) in the frequency of differentiating F3-4 from F0-2 fibrosis. Only hepatic nodularity and PVH stigmata showed moderately high overall accuracy of 69.4% and 72.2%. Interreader agreement was substantial only for PVH stigmata, moderate for C/R m, deep notch, and expanded gallbladder fossa. Only posterior hepatic notch (P = 0.82) showed no significant difference in reader rating.

CONCLUSIONS

MRE is a noninvasive, accurate, and reproducible technique compared with conventional features of detecting severe hepatic fibrosis.

This report describes 2 patients with a clinical and hematologic diagnosis of chronic myeloid leukemia (CML) in chronic phase who had an acquired t(8;22)(p11;q11). Analysis by fluorescence in situ hybridization (FISH) and reverse transcription-polymerase chain reaction (RT-PCR) indicated that both patients were negative for the BCR-ABL fusion, but suggested that the BCR gene was disrupted. Further FISH indicated a breakpoint within fibroblast growth factor receptor 1 (FGFR1), the receptor tyrosine kinase that is known to be disrupted in a distinctive myeloproliferative disorder, most commonly by fusion to ZNF198. RT-PCR confirmed the presence in both cases of an in-frame messenger RNA fusion between BCR exon 4 and FGFR1 exon 9. Expression of BCR-FGFR1 in the factor-dependent cell line Ba/F3 resulted in interleukin 3-independent clones that grew at a comparable rate to cells transformed with ZNF198-FGFR1. The growth of transformed cells was inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002, the farnesyltransferase inhibitors L744832 and manumycin A, the p38 inhibitors SB202190 and SB203580 but not by the MEK inhibitor PD98059. The growth of BaF3/BCR-FGFR1 and BaF3/ZNF198-FGFR1 was not significantly inhibited by treatment with STI571, but was inhibited by SU5402, a compound with inhibitory activity against FGFR1. Inhibition with this compound was associated with decreased phosphorylation of ERK1/2 and BCR-FGFR1 or ZNF198-FGFR1, and was dose dependent with an inhibitory concentration of 50% of approximately 5 microM. As expected, growth of BaF3/BCR-ABL was inhibited by STI571 but not by SU5402. The study demonstrates that the BCR-FGFR1 fusion may occur in patients with apparently typical CML. Patients with constitutively active FGFR1 fusion genes may be amenable to treatment with specific FGFR1 inhibitors.

In comparison with essential hypertension, primary aldosteronism (PA) is associated with an increased risk of cardiovascular morbidity. To date, no data on mortality have been published. We assessed mortality of patients treated for PA within the German Conn's registry and identified risk factors for adverse outcome in a case-control study. Patients with confirmed PA treated in 3 university centers in Germany since 1994 were included in the analysis. All of the patients were contacted in 2009 and 2010 to verify life status. Subjects from the population-based F3 survey of the Cooperative Health Research in the Region of Augsburg served as controls. Final analyses were based on 600 normotensive controls, 600 hypertensive controls, and 300 patients with PA. Kaplan-Meyer survival curves were calculated for both cohorts. Ten-year overall survival was 95% in normotensive controls, 90% in hypertensive controls, and 90% in patients with PA (P value not significant). In multivariate analysis, age (hazard ratio, 1.09 per year [95% CI, 1.03-1.14]), angina pectoris (hazard ratio, 3.6 [95% CI, 1.04-12.04]), and diabetes mellitus (hazard ratio, 2.55 [95% CI, 1.07-6.09]) were associated with an increase in all-cause mortality, whereas hypokalemia (hazard ratio, 0.41 per mmol/L [95% CI, 0.17-0.99]) was associated with reduced mortality. Cardiovascular mortality was the main cause of death in PA (50% versus 34% in hypertensive controls; P<0.05). These data indicate that cardiovascular mortality is increased in patients treated for PA, whereas all-cause mortality is not different from matched hypertensive controls.

OBJECTIVE

Hallux valgus is a common orthopaedic condition affecting elderly people. Grading the severity of the condition commonly involves obtaining measurements from radiographs, which may not be feasible or necessary in some clinical or research settings. Recently, a non-invasive clinical assessment tool (the Manchester scale), consisting of four standardized photographs, has been developed; however, its validity has not yet been determined. Therefore, the objective of this study was to determine the validity of this tool by correlating Manchester scale scores with hallux valgus measurements obtained from radiographs.

METHODS

Weight-bearing dorsoplantar foot radiographs were obtained from 95 subjects (31 men and 64 women) aged 62-94 yr (mean 78.6, s.d. 6.5), and measurements of the hallux abductus angle, intermetatarsal angle and hallux interphalangeal adbuctus angle were performed. These measurements were then correlated with the Manchester scale scores (none, mild, moderate or severe).

RESULTS

The Manchester scale score was highly correlated with hallux abductus angle (Spearman's rho = 0.73, P<0.01) and moderately associated with intermetatarsal angle (rho = 0.49, P<0.01) measurements obtained from radiographs. Analysis of variance revealed significant differences in mean hallux abductus angles [F3 = 119.99, P<0.001] and intermetatarsal angles [F3 =29.56, P<0.001] between the four Manchester scale categories.

CONCLUSIONS

These findings indicate that the Manchester scale provides a valid representation of the degree of hallux valgus deformity determined from radiographic measurement of hallux abductus angle and intermetatarsal angle. We therefore recommend the use of this instrument as a simple, non-invasive screening tool for clinical and research purposes.

Fifteen subjects were presented with series of tones. Any one tone was either loud or soft, and in any one series the probability of one tone intensity was either 0.9 or 0.1. Subjects were instructed to count the frequent tones or to count the rare tones. The stimuli were also presented while the subjects were solving a word-puzzle. Event-related potentials (ERPs) were recorded from 9 scalp locations (F3, C3, P3, FZ, CZ, PZ, F4, C4, P4) referred to linked mastoids. ERP components were measured with a Principal Components analysis and the relations between these measures and the independent variables were evaluated with the ANOVA procedure. This paradigm allowed an evaluation of the effect of stimulus probability, stimulus relevance, and task relevance on the waveform of the ERPs. We conclude that the P350 component is enhanced whenever the eliciting stimulus is both rare and in some sense relevant to the subject's task and the degree of enhancement is greatest when the rare--relevant tone is loud. A "slow wave" component which follows P350 is related to the same variables but has a scalp distribution quite different from that of P350. The slow wave shows a progressive shift in polarity from negative to positive from the frontal to the parietal sites, while the P350 is of nearly equal amplitude (and positive) at the central and parietal sites and has a smaller (positive) and amplitude at FZ. A third prominent component, negative in polarity, peaking at about 210 msec, is most pronounced following rare stimuli, whether or not they were task relevant. The amplitude of N210 tended to be largest at the frontal electrode. This study then demonstrates that when suitable measurement techniques are used, multiple endogenous ERP components can be observed, each related to distinct aspects of cognitive behavior.

The IL-2R promotes rapid expansion of activated T cells through signals mediated by the adaptor protein Shc and the transcription factor Stat5. The mechanisms that engage the cell cycle are not well defined. We report on the transcriptional regulation of the cell cycle gene cyclin D2 by the IL-2R. IL-2-responsive induction of a luciferase reporter gene containing 1624 bp of the cyclin D2 promoter/enhancer was studied in the murine CD8(+) T cell line CTLL2. Reporter gene deletional analysis and EMSAs indicate an IL-2-regulated enhancer element flanks nucleotide -1204 and binds a complex of at least three proteins. The enhancer element is bound constitutively by Sp1 and an unknown factor(s) and inducibly by Stat5 in response to IL-2. The Stat5 binding site was essential for IL-2-mediated reporter gene activity, and maximum induction required the adjacent Sp1 binding site. Receptor mutagenesis studies in the pro-B cell line BA/FG (a derivative of the BA/F3 cell line) demonstrated a correlation between Stat5 activity and cyclin D2 mRNA levels when the Stat5 signal was isolated, disrupted, and then rescued. Further, a dominant-negative form of Stat5 lacking the trans-activation domain inhibited induction of cyclin D2 mRNA. We propose that the IL-2R regulates the cyclin D2 gene in part through formation of an enhancer complex containing Stat5 and Sp1.

Myosin X (MyoX), encoded by Myo10, is a representative member of the MyTH4-FERM domain-containing myosins, and this family of unconventional myosins shares common functions in promoting formation of filopodia/stereocilia structures in many cell types with unknown mechanisms. Here, we present the structure of the MyoX MyTH4-FERM tandem in complex with the cytoplasmic tail P3 domain of the netrin receptor DCC. The structure, together with biochemical studies, reveals that the MyoX MyTH4 and FERM domains interact with each other, forming a structural and functional supramodule. Instead of forming an extended β-strand structure in other FERM binding targets, DCC_P3 forms a single α-helix and binds to the αβ-groove formed by β5 and α1 of the MyoX FERM F3 lobe. Structure-based amino acid sequence analysis reveals that the key polar residues forming the inter-MyTH4/FERM interface are absolutely conserved in all MyTH4-FERM tandem-containing proteins, suggesting that the supramodular nature of the MyTH4-FERM tandem is likely a general property for all MyTH4-FERM proteins.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) are members of the interleukin-6 (IL-6) subfamily of cytokines that use a common signal transducer gp130. Human OSM (hOSM) and LIF share a functional high-affinity receptor that is composed of gp130 and LIF receptor beta subunit (LIFRbeta). A second high-affinity receptor for hOSM was recently found to be formed by gp130 and the hOSM receptor beta subunit. However, the nature of murine OSM (mOSM) and its receptors has remained unknown. Using the recently cloned mOSM cDNA, we produced recombinant mOSM and studied its biological activity and receptor structure. Murine hematopoietic cell lines M1 and DA1.a, an embryonic stem cell line CCE, and Ba/F3 transfectants expressing gp130 and LIFRbeta responded to murine LIF (mLIF) and hOSM equally well, while these cells responded to mOSM only at a 30-fold to 100-fold higher concentration than those of mLIF and hOSM. In contrast, NIH3T3 cells responded to mOSM, but not to mLIF and hOSM. Scatchard plot analyses showed that mOSM bound to gp130 with low-affinity (kd = 2.8 to 4.2 nmol/L) and that the binding affinity did not increase in the presence of LIFRbeta. However, mOSM bound to NIH3T3 cells with high-affinity (kd = 660 pmol/L), whereas mLIF did not bind to NIH3T3 cells at all. These results indicate that unlike hOSM, mOSM and mLIF do not share the same functional receptor, and mOSM delivers signals only through its specific receptor complex. Further studies in mice will define the physiological roles of OSM.

The development of a drug delivery strategy which can mediate efficient tumor targeting together with high cellular internalization and extensive vascular extravasation is essential and important for glioma treatment. To achieve this goal, F3 peptide that specifically bind to nucleolin, which is highly expressed on the surface of both glioma cells and endothelial cells of glioma angiogenic blood vessels, is utilized to decorate a nanoparticulate drug delivery system to realize glioma cell and neovasculature dual-targeting and efficient cellular internalization. Tumor homing and penetrating peptide, tLyp-1 peptide, which contains the motif of (R/K)XX(R/K) and specially binds to neuropilin is co-administrated to improve the penetration of the nanoparticles across angiogenic vasculature into glioma parenchyma. The F3 conjugation via a maleimide-thiol coupling reaction was confirmed by XPS analysis with 1.03% nitrogen detected on the surface of the functionalized nanoparticles. Enhanced cellular interaction with C6 cells, improved penetration in 3D multicell tumor spheroids, and increased cytotoxicity of the loaded paclitaxel were achieved by the F3-functionalized nanoparticles (F3-NP). Following co-administration with tLyp-1 peptide, F3-NP displayed enhanced accumulation at the tumor site and deep penetration into the glioma parenchyma and achieved the longest survival in mice bearing intracranial C6 glioma. The findings here clearly indicated that the strategy by co-administrating a tumor homing and penetrating peptide with functionalized nanoparticles dual-targeting both glioma cells and neovasculature could significantly improve the anti-glioma drug delivery, which also hold a great promise for chemotherapy of other hard-to-cure cancers.

c-Mpl, a member of the hematopoietic cytokine receptor family, is the receptor for thrombopoietin. To investigate signal transduction by c-Mpl, a chimeric receptor, composed of the extracellular domain of human growth hormone receptor and the intracellular domain of c-Mpl, was introduced into the interleukin 3-dependent cell line Ba/F3. In response to growth hormone, this chimeric receptor induced growth in the absence of interleukin 3. Deletion analysis of the 123-amino acid intracellular domain indicated that the elements responsible for this effect are present within the 63 amino acids proximal to the transmembrane domain. Mutation of the recently described box 1 motif abrogated the proliferative response. Tyrosine phosphorylation of the tyrosine kinase JAK-2 and activation of STAT proteins were dependent on box 1 and sequences within 63 amino acids of the plasma membrane. STAT proteins activated by thrombopoietin in a megakaryocytic cell line were purified and shown to be STAT1 and STAT3. A separate region located at the C terminus of the c-Mpl intracellular domain was found to be required for induction of Shc phosphorylation and c-fos mRNA accumulation, suggesting involvement of the Ras signal transduction pathway. Thus, at least two distinct regions are involved in signal transduction by the c-Mpl.

We have utilized site-directed mutants to study the role of autophosphorylation of the epidermal growth factor (EGF) receptor in the regulation of receptor kinase activity and ligand-induced endocytosis. A single mutation of the major autophosphorylation site, Y1173, and a double mutation of two autophosphorylation sites, Y1173 and Y1148, did not inhibit kinase activity in vivo, using PLC gamma 1 as a specific substrate for the EGF receptor kinase. The simultaneous mutation of three major autophosphorylation sites (Y1173, Y1148, Y1068), however, caused more than a 50% decrease in EGF-induced tyrosine phosphorylation of PLC gamma 1. The triple mutation also resulted in a substantial inhibition of the EGF-receptor endocytic system. We have used three types of experiments to analyze internalization, recycling, and degradation of EGF in cells with these mutants or the wild-type receptor. Using a simple mathematical model we have shown that the internalization rate constant is 2-fold lower in cells expressing the triple mutation receptor (F3 cells) than in cells expressing wild-type EGF receptor (wild-type cells). However, the rate constant for recycling was similar in both cell types. The EGF degradation rate constant was also lower in F3 cells. EGF-induced EGF receptor degradation was slower in F3 cells (t1/2 = 4 h) than in wild-type cells (t1/2 = 1 h). Therefore, our results suggest that multiple autophosphorylations of the carboxyl terminus of the EGF receptor are required for EGF receptor kinase activation, and for the internalization and intracellular processing of the EGF.receptor complex.

We report the fusion of the Huntingtin interactin protein 1 (HIP1) gene to the platelet-derived growth factor betareceptor (PDGFbetaR) gene in a patient with chronic myelomonocytic leukemia (CMML) with a t(5;7)(q33;q11.2) translocation. Southern blot analysis of patient bone marrow cells with a PDGFbetaR gene probe demonstrated rearrangement of the PDGFbetaR gene. Anchored polymerase chain reaction using PDGFbetaR primers identified a chimeric transcript containing the HIP1 gene located at 7q11.2 fused to the PDGFbetaR gene on 5q33. HIP1 is a 116-kD protein recently cloned by yeast two-hybrid screening for proteins that interact with Huntingtin, the mutated protein in Huntington's disease. The consequence of t(5;7)(q33;q11.2) is an HIP1/PDGFbetaR fusion gene that encodes amino acids 1 to 950 of HIP1 joined in-frame to the transmembrane and tyrosine kinase domains of the PDGFbetaR. The reciprocal PDGFbetaR/HIP1 transcript is not expressed. HIP1/PDGFbetaR is a 180-kD protein when expressed in the murine hematopoietic cell line, Ba/F3, and is constitutively tyrosine phosphorylated. Furthermore, HIP1/PDGFbetaR transforms the Ba/F3 cells to interleukin-3-independent growth. These data are consistent with an alternative mechanism for activation of PDGFbetaR tyrosine kinase activity by fusion with HIP1, leading to transformation of hematopoietic cells, and may implicate Huntingtin or HIP1 in the pathogenesis of hematopoietic malignancies.

BACKGROUND

HCV protease inhibitors (PIs) boceprevir and telaprevir in combination with PEG-Interferon alfa and Ribavirin (P/R) is the new standard of care in the treatment of chronic HCV genotype 1 (GT1) infection. However, not every HCV GT1 infected patient is eligible for P/R/PI therapy. Furthermore phase III studies did not necessarily reflect real world as patients with advanced liver disease or comorbidities were underrepresented. The aim of our study was to analyze the eligibility and safety of P/R/PI treatment in a real world setting of a tertiary referral center.

METHODS

All consecutive HCV GT1 infected patients who were referred to our hepatitis treatment unit between June and November 2011 were included. Patients were evaluated for P/R/PI according to their individual risk/benefit ratio based on 4 factors: Treatment-associated safety concerns, chance for SVR, treatment urgency and nonmedical patient related reasons. On treatment data were analyzed until week 12.

RESULTS

208 patients were included (F3/F4 64%, mean platelet count 169/nl, 40% treatment-naïve). Treatment was not initiated in 103 patients most frequently due to safety concerns. 19 patients were treated in phase II/III trials or by local centers and a triple therapy concept was initiated at our unit in 86 patients. Hospitalization was required in 16 patients; one patient died due to a gastrointestinal infection possibly related to treatment. A platelet count of <110/nl was associated with hospitalization as well as treatment failure. Overall, 128 patients were either not eligible for therapy or experienced a treatment failure at week 12.

CONCLUSIONS

P/R/PI therapies are complex, time-consuming and sometimes dangerous in a real world setting, especially in patients with advanced liver disease. A careful patient selection plays a crucial role to improve safety of PI based therapies. A significant number of patients are not eligible for P/R/PI, emphasizing the need for alternative therapeutic options.

BACKGROUND

Chronic stress is considered to be one of many causes of human preterm birth (PTB), but no direct evidence has yet been provided. Here we show in rats that stress across generations has downstream effects on endocrine, metabolic and behavioural manifestations of PTB possibly via microRNA (miRNA) regulation.

METHODS

Pregnant dams of the parental generation were exposed to stress from gestational days 12 to 18. Their pregnant daughters (F1) and grand-daughters (F2) either were stressed or remained as non-stressed controls. Gestational length, maternal gestational weight gain, blood glucose and plasma corticosterone levels, litter size and offspring weight gain from postnatal days 1 to 30 were recorded in each generation, including F3. Maternal behaviours were analysed for the first hour after completed parturition, and offspring sensorimotor development was recorded on postnatal day (P) 7. F0 through F2 maternal brain frontal cortex, uterus and placenta miRNA and gene expression patterns were used to identify stress-induced epigenetic regulatory pathways of maternal behaviour and pregnancy maintenance.

RESULTS

Progressively up to the F2 generation, stress gradually reduced gestational length, maternal weight gain and behavioural activity, and increased blood glucose levels. Reduced offspring growth and delayed behavioural development in the stress cohort was recognizable as early as P7, with the greatest effect in the F3 offspring of transgenerationally stressed mothers. Furthermore, stress altered miRNA expression patterns in the brain and uterus of F2 mothers, including the miR-200 family, which regulates pathways related to brain plasticity and parturition, respectively. Main miR-200 family target genes in the uterus, Stat5b, Zeb1 and Zeb2, were downregulated by multigenerational stress in the F1 generation. Zeb2 was also reduced in the stressed F2 generation, suggesting a causal mechanism for disturbed pregnancy maintenance. Additionally, stress increased placental miR-181a, a marker of human PTB.

CONCLUSIONS

The findings indicate that a family history of stress may program central and peripheral pathways regulating gestational length and maternal and newborn health outcomes in the maternal lineage. This new paradigm may model the origin of many human PTB causes.

The epidermal growth factor (EGF) family hormones amphiregulin (AR), transforming growth factor-alpha (TGF-alpha), and heparin-binding EGF-like growth factor (HB-EGF) are thought to play significant roles in the genesis or progression of a number of human malignancies. However, the ability of these ligands to activate all four erbB family receptors has not been evaluated. Therefore, we have assessed the stimulation of erbB family receptor tyrosine phosphorylation by these hormones in a panel of mouse Ba/F3 cell lines expressing the four erbB family receptors, singly and in pairwise combinations. We also measured the stimulation of interleukin-3-independent survival or proliferation in this panel of Ba/F3 cell lines to compare the patterns of erbB family receptor coupling to physiologic responses induced by these peptides. EGF, TGF-alpha, AR, and HB-EGF all stimulated qualitatively similar patterns of erbB family receptor tyrosine phosphorylation and coupling to physiologic responses. Therefore, EGF, TGF-alpha, AR, and HB-EGF are functionally identical in this model system and behave differently from the EGF family hormones betacellulin and neuregulins.