The comprehensive genetic alterations underlying the pathogenesis of T-cell prolymphocytic leukemia (T-PLL) are unknown. To address this, we performed whole-genome sequencing (WGS), whole-exome sequencing (WES), high-resolution copy-number analysis, and Sanger resequencing of a large cohort of T-PLL. WGS and WES identified novel mutations in recurrently altered genes not previously implicated in T-PLL including EZH2, FBXW10, and CHEK2. Strikingly, WGS and/or WES showed largely mutually exclusive mutations affecting IL2RG, JAK1, JAK3, or STAT5B in 38 of 50 T-PLL genomes (76.0%). Notably, gain-of-function IL2RG mutations are novel and have not been reported in any form of cancer. Further, high-frequency mutations in STAT5B have not been previously reported in T-PLL. Functionally, IL2RG-JAK1-JAK3-STAT5B mutations led to signal transducer and activator of transcription 5 (STAT5) hyperactivation, transformed Ba/F3 cells resulting in cytokine-independent growth, and/or enhanced colony formation in Jurkat T cells. Importantly, primary T-PLL cells exhibited constitutive activation of STAT5, and targeted pharmacologic inhibition of STAT5 with pimozide induced apoptosis in primary T-PLL cells. These results for the first time provide a portrait of the mutational landscape of T-PLL and implicate deregulation of DNA repair and epigenetic modulators as well as high-frequency mutational activation of the IL2RG-JAK1-JAK3-STAT5B axis in the pathogenesis of T-PLL. These findings offer opportunities for novel targeted therapies in this aggressive leukemia.

Drought is an important climatic phenomenon which, after soil infertility, ranks as the second most severe limitation to maize production in developing countries. When drought stress occurs just before or during the flowering period, a delay in silking is observed, resulting in an increase in the length of the anthesis-silking interval (ASI) and in a decrease in grain yield. Selection for reduced ASI in tropical open-pollinated varieties has been shown to be correlated with improved yields under drought stress. Since efficient selection for drought tolerance requires carefully managed experimental conditions, molecular markers were used to identify the genomic segments responsible for the expression of ASI, with the final aim of developing marker-assisted selection (MAS) strategies. An F2population of 234 individuals was genotyped at 142 loci and F3 families were evaluated in the field under several water regimes for male flowering (MFLW), male sterility (STER), female flowering (FFLW) and ASI. The genetic variance of ASI increased as a function of the stress intensity, and the broad-sense heritabilites of MFLW, FFLW and ASI were high under stress conditions, being 86%, 82% and 78%, respectively. Putative quantitative trait loci (QTLs) involved in the expression of MFLW and/or FFLW under drought were detected on chromosomes 1, 2, 4, 5, 8, 9 and 10, accounting for around 48% of the phenotypic variance for both traits. For ASI, six putative QTLs were identified under drought on chromosomes 1, 2, 5, 6, 8 and 10, and together accounted for approximately 47% of the phenotypic variance. Under water stress conditions, four QTLs were common for the expression of MFLW and FFLW, one for the expression of ASI and MFLW, and four for the expression of ASI and FFLW. The number of common QTLs for two traits was related to the level of linear correlation between these two traits. Segregation for ASI was found to be transgressive with the drought-susceptible parent contributing alleles for reduced ASI (4 days) at two QTL positions. Alleles contributed by the resistant line at the other four QTLs were responsible for a 7-day reduction of ASI. These four QTLs represented around 9% of the linkage map, and were stable over years and stress levels. It is argued that MAS based on ASI QTLs should be a powerful tool for improving drought tolerance of tropical maize inbred lines.

Neurons and glia in the vertebrate central nervous system arise in temporally distinct, albeit overlapping, phases. Neurons are generated first followed by astrocytes and oligodendrocytes from common progenitor cells. Increasing evidence indicates that axon-derived signals spatiotemporally modulate oligodendrocyte maturation and myelin formation. Our previous observations demonstrate that F3/contactin is a functional ligand of Notch during oligodendrocyte maturation, revealing the existence of another group of Notch ligands. Here, we establish that NB-3, a member of the F3/contactin family, acts as a novel Notch ligand to participate in oligodendrocyte generation. NB-3 triggers nuclear translocation of the Notch intracellular domain and promotes oligodendrogliogenesis from progenitor cells and differentiation of oligodendrocyte precursor cells via Deltex1. In primary oligodendrocytes, NB-3 increases myelin-associated glycoprotein transcripts. Thus, the NB-3/Notch signaling pathway may prove to be a molecular handle to treat demyelinating diseases.

Apoptotic cells expose phosphatidylserine on their surface as an "eat me" signal, and macrophages respond by engulfing them. Although several molecules that specifically bind phosphatidylserine have been identified, the molecular mechanism that triggers engulfment remains elusive. Here, using a mouse pro-B cell line, Ba/F3, that grows in suspension, we reconstituted the engulfment of apoptotic cells. The parental Ba/F3 cells did not engulf apoptotic cells. Ba/F3 transformants expressing T cell immunoglobulin- and mucin-domain-containing molecule 4 (Tim4), a type I membrane protein that specifically binds phosphatidylserine, efficiently bound apoptotic cells in a phosphatidylserine-dependent manner but did not engulf them. However, Ba/F3 transformants expressing both Tim4 and the integrin α(v)β(3) complex bound to and engulfed apoptotic cells in the presence of milk fat globule epidermal growth factor factor VIII (MFG-E8), a secreted protein that can bind phosphatidylserine and integrin α(v)β(3). These results indicate that the engulfment of apoptotic cells proceeds in two steps: Tim4 tethers apoptotic cells, and the integrin α(v)β(3) complex mediates engulfment in coordination with MFG-E8. A similar two-step engulfment of apoptotic cells was observed with mouse resident peritoneal macrophages. Furthermore, the Tim4/integrin-mediated engulfment by the Ba/F3 cells was enhanced in cells expressing Rac1 and Rab5, suggesting that this system well reproduces the engulfment of apoptotic cells by macrophages.

OBJECTIVE

The objective of our study was to determine the usefulness of the apparent diffusion coefficient (ADC) of liver parenchyma for determining the severity of liver fibrosis.

METHODS

This study investigated 78 patients who underwent diffusion-weighted imaging (DWI) with 1.5-T MRI and pathologic staging of liver fibrosis based on biopsy. DWI was performed with b values of 50 and 400 s/mm(2). ADCs of liver were measured using 2.0- to 3.0-cm(2) regions of interest in the right and left lobes of the liver; the mean ADC value was used for analysis. Pathologic METAVIR scores for liver fibrosis stage were used as a reference standard.

RESULTS

The mean ADC values for fibrosis pathologically staged using the METAVIR classification system as F0 (n = 11), F1 (n = 16), F2 (n = 10), F3 (n = 14), and F4 (n = 27) were 125.9, 105.0, 104.5, 103.2, and 99.1 x 10(-5) s/mm(2), respectively. The correlation between the ADC values and the degree of liver fibrosis was moderate (Spearman's test, rho = -0.36). There was a significant difference in ADC values between patients with nonfibrotic liver (F0) and those with cirrhotic liver (F4) (p = 0.008). The best cutoff ADC value to distinguish between these groups was 118 x 10(-5) s/mm(2). However, ADC values were not useful for differentiating viral hepatitis patients with F2 fibrosis or higher from those with a lower degree of fibrosis (area under the receiver operating characteristic curve [AUC] = 0.66) or for differentiating low-stage fibrosis in all patients from high-stage fibrosis in all patients (AUC = 0.54).

CONCLUSIONS

The ADCs in cirrhotic livers are significantly lower than those in nonfibrotic livers. However, ADC values measured using the current generation of scanners are not reliable enough to replace liver biopsy for staging hepatic fibrosis.

The proto-oncogene KIT encodes the receptor tyrosine kinase KIT. Gain-of-function mutations in the juxtamembrane domain of KIT have been reported in human gastrointestinal stromal tumors. In a family with multiple gastrointestinal stromal tumors and diffuse hyperplasia of myenteric plexus layer, we have identified another mutation of KIT, a single base mutation, resulting in the substitution of Glu for Lys(642) in the kinase I domain, and studied its biological effect in a cellular system. The mouse homologue of the human KIT mutant was generated by site-directed mutagenesis and stably transfected into the interleukin-3-dependent Ba/F3 murine cell line. The oncogenic potential of the mutated KIT was assessed in vitro by a proliferation assay and in vivo by transplantation into nude mice. Transfected Ba/F3 cells grew autonomously in absence of growth factors and formed tumors in nude mice. Substitution of Glu for Lys(642) is an oncogenic mutation in the tyrosine kinase domain of KIT. As germline heterozygous mutation, it causes a diffuse hyperplasia of myenteric interstitial cells of Cajal during embryonic development and occurrence of multiple gastrointestinal stromal tumors at adulthood.

Sixteen different mature interspecific parasexual hybrids, produced by fusing leaf protoplasts of Nicotiana glauca (G) and N. langsdorffii (L), were analyzed for fraction I protein (ribulose-1,5-bisphosphate carboxylase/oxygenase) which consists of large subunit polypeptides coded by chloroplast DNA and small subunit polypeptides coded by nuclear DNA. All the hybrids showed the combined small subunits of both parents, thus confirming the hybridity of each of the fusion products. Fourteen of the hybrids displayed the large subunit electrofocusing pattern characteristic of only one parent (eight L and six G). From one hybrid callus, two plants were regenerated, of which one had exclusively L-type large subunit and the other had exclusively G. A single plant retained a mixture of L ang G chloroplast DNA's; this later yielded six F2 progeny from one branch, all of which were G type, and three asexual progeny from another branch, all of which had the L-type pattern. In all, 46 F2 progeny and 8 different F3s were analyzed and each of these, with few if any exceptions, showed the same single subunit type as the F1 and F2 parent hybrid plants. Reasons for the rapid sorting out of the chloroplast types are discussed.

STAT5a and STAT5b are two highly related transcription factors that control essential cellular functions. Several STAT5 targets are known, although it is likely that most remain uncharacterized. To identify a more complete set of STAT5-regulated genes, we used a modification of the chromatin immunoprecipitation procedure, which does not presuppose any information regarding these targets. Employing Ba/f3 cells in which STAT5 is activated by interleukin-3, we have identified novel STAT5 binding sites that may be regulatory regions for nearby genes. These sites are typically found far from transcription start sites, and most do not contain CpG islands, indicating that they are not in traditional promoter regions. Nonetheless, when the expression of genes near these STAT5 binding sites was examined, all were expressed in Ba/f3 cells, and most were modulated by interleukin-3. Furthermore, genes identified by this strategy show unique expression patterns in acute leukemias, tumors characterized by activated STAT5. Whereas both STAT5 isoforms bound to all promoters tested, STAT5a and STAT5b bound with different kinetics, suggesting that at least some of the differences between the functions of these two proteins are mediated by their DNA binding activity. Therefore, this method of transcription factor target identification represents an effective strategy to isolate transcription factor targets in an unbiased fashion, and it has revealed many novel STAT5-dependent regulatory regions outside of traditional promoters.

Endoscopic observations of gastric varices in 124 patients were classified according to form, location, and color. Form was classified into three types: tortuous (F1), nodular (F2), and tumorous (F3). Location was classified into five types: anterior (La), posterior (Lp), lesser (Ll) and greater curvature (Lg) of the cardia, and fundic area (Lf). Color was white (Cw) or red (Cr). Glossy, thin-walled focal redness on the varix was defined as red color spot (RC spot). Stepwise logistic regression analysis for multivariate adjustments was performed for all of the endoscopic covariates, and four risk factors (La, Lg, F2, RC spot) that affect bleeding from gastric varices were independently identified. This classification should aid in assessing gastric varices observed by fiberoptic endoscopy and help design appropriate treatment.

Plasmodium vivax is highly endemic in the lowlands of Papua New Guinea and accounts for a large proportion of the malaria cases in children less than 5 years of age. We collected 2117 blood samples at 2-monthly intervals from a cohort of 268 children aged 1 to 4.5 years and estimated the diversity and multiplicity of P. vivax infection. All P. vivax clones were genotyped using the merozoite surface protein 1 F3 fragment (msp1F3) and the microsatellite MS16 as molecular markers. High diversity was observed with msp1F3 (H(E) = 88.1%) and MS16 (H(E) = 97.8%). Of the 1162 P. vivax positive samples, 74% harbored multi-clone infections with a mean multiplicity of 2.7 (IQR = 1-3). The multiplicity of P. vivax infection increased slightly with age (P = 0.02), with the strongest increase in very young children. Intensified efforts to control malaria can benefit from knowledge of the diversity and MOI both for assessing the endemic situation and monitoring the effects of interventions.

The Wisteria floribunda agglutinin-positive human Mac-2-binding protein (WFA+-M2BP) was recently shown to be a liver fibrosis glycobiomarker with a unique fibrosis-related glycoalteration. We evaluated the ability of WFA+-M2BP to predict the development of hepatocellular carcinoma (HCC) in patients who were infected with the hepatitis C virus (HCV). A total of 707 patients who had been admitted to our hospital with chronic HCV infection without other potential risk factors were evaluated to determine the ability of WFA+-M2BP to predict the development of HCC; factors evaluated included age, sex, viral load, genotypes, fibrosis stage, aspartate and alanine aminotransferase levels, bilirubin, albumin, platelet count, alpha-fetoprotein (AFP), WFA+-M2BP, and the response to interferon (IFN) therapy. Serum WFA+-M2BP levels were significantly increased according to the progression of liver fibrosis stage (P<0.001). In each distinctive stage of fibrosis (F0-F1, F2, F3, and F4), the risk of development of HCC was increased according to the elevation of WFA+-M2BP. Multivariate analysis identified age>57 years, F4, AFP>20 ng/mL, WFA+-M2BP ≥4, and WFA+-M2BP 1-4 as well as the response to IFN (no therapy vs. sustained virological response) as independent risk factors for the development of HCC. The time-dependent areas under the receiver operating characteristic curve demonstrated that the WFA+-M2BP assay predicted the development of HCC with higher diagnostic accuracy than AFP.

CONCLUSIONS

WFA+-M2BP can be applied as a useful surrogate marker for the risk of HCC development, in addition to liver biopsy.

Herpes simplex virus type 1 (HSV-1) B capsids are composed of seven proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26 in order of decreasing molecular weight. Three proteins (21, 22a, and VP24) are encoded by a single open reading frame (ORF), UL26, and include a protease whose structure and function have been studied extensively by other investigators. The protease encoded by this ORF generates VP24 (amino acids 1 to 247), a structural component of the capsid and mature virions, and 21 (residues 248 to 635). The protease also cleaves C-terminal residues 611 to 635 of 21 and 22a, during capsid maturation. Protease activity has been localized to the N-terminal 247 residues. Protein 22a and probably the less abundant protein 21 occupy the internal volume of capsids but are not present in virions; therefore, they may form a scaffold that is used for B capsid assembly. The objective of the present study was to isolate and characterize a mutant virus with a null mutation in UL26. Vero cells were transformed with plasmid DNA that encoded ORF UL25 through UL28 and screened for their ability to support the growth of a mutant virus with a null mutation in UL27 (K082). Four of five transformants that supported the growth of the UL27 mutant also supported the growth of a UL27-UL28 double mutant. One of these transformants (F3) was used to isolate a mutant with a null mutation in UL26. The UL26 null mutation was constructed by replacement of DNA sequences specifying codons 41 through 593 with a lacZ reporter cassette. Permissive cells were cotransfected with plasmid and wild-type virus DNA, and progeny viruses were screened for their ability to grow on F3 but not Vero cells. A virus with these growth characteristics, designated KUL26 delta Z, that did not express 21, 22a, or VP24 during infection of Vero cells was isolated. Radiolabeled nuclear lysates from infected nonpermissive cells were layered onto sucrose gradients and subjected to velocity sedimentation. A peak of radioactivity for KUL26 delta Z that sedimented more rapidly than B capsids from wild-type-infected cells was observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the gradient fractions showed that the peak fractions contained VP5, VP19C, VP23, and VP26. Analysis of sectioned cells and of the peak fractions of the gradients by electron microscopy revealed sheet and spiral structures that appear to be capsid shells.(ABSTRACT TRUNCATED AT 400 WORDS)

Patients with advanced stages of chronic myeloid leukemia (CML) often manifest imatinib mesylate resistance associated with point mutations in BCR-ABL. AMN107 is a new higher-potency inhibitor of BCR-ABL. To identify mutations in BCR-ABL that could result in resistance to AMN107, a cDNA library of BCR-ABL mutants was introduced into Ba/F3 cells followed by selection in AMN107 (0.125-0.5 microM). A total of 86 individual, drug-resistant colonies were recovered, and the SH3, SH2, and kinase domains of BCR-ABL were sequenced. A total of 46 colonies had single point mutations in BCR-ABL, with a total of 17 different mutations, all within the kinase domain. The other 40 colonies had multiple point mutations and were not analyzed further. Each of the 17 single point mutants were reconstructed by site-directed mutagenesis of native BCR-ABL and found to be approximately 2.5- to 800-fold more resistant to AMN107 than native BCR-ABL. The mutations included 6 known imatinib mesylate-resistant mutations, including T315I, which showed complete resistance to AMN107. Interestingly, most AMN107-resistant mutants were also resistant to imatinib mesylate. These results may predict some of the resistance mutations that will be detected in clinical trials with this kinase inhibitor.

An effort has been made to integrate insights on charge-based interactions in immune complex glomerulonephritis with nuclear antigen involvement in lupus nephritis. Attention was focussed on the histones, a group of highly cationic nuclear constituents, which could be expected to bind to fixed anionic sites present in the glomerular basement membrane (GBM). We demonstrated that all histone subfractions, prepared according to Johns (4), have a high affinity for GBM and the basement membrane of peritubular capillaries. Tissue uptake of 125I-labeled histones was measured by injecting 200 micrograms of each fraction into the left kidney via the aorta and measuring organ uptake after 15 min. In glomeruli isolated from the left kidneys, the following quantities of histones were found: f1, 13 micrograms; f2a (f2al + f2a2), 17 micrograms; f2b, 17 micrograms; and f3, 32 micrograms. Kinetic studies of glomerular binding showed that f1 disappeared much more rapidly than f2a. The high affinity of histones (pI between 10.5 and 11.0; mol wt 10,000-22,000) for the GBM correlates well with their ability to form aggregates (mol wt greater than 100,000) for comparison lysozyme (pI 11, mol wt 14,000), which does not aggregate spontaneously bound poorly (0.4 micrograms in isolated glomeruli). The quantity of histones and lysozyme found in the isolated glomeruli paralleled their in vitro affinity for a Heparin-Sepharose column (gradient elution studies). This gel matrix contains the sulfated, highly anionic polysaccharide heparin, which is similar to the negatively charged heparan sulfate present in the GBM. Lysozyme eluted with 0.15 M NaCl, f1 with 1 M NaCl, and f2a, f2b, and f3 could not be fully desorbed even with 2 M NaCl; 6 M guanidine-HCl was necessary. Two further findings of great relevance for the concept of induction of immune complex glomerulonephritis by histones were: (a) glomerular-bound histone was accessible for specific antibody given intravenously; and (b) prior binding of histones promoted glomerular deposition of anionic antigens, as could be shown with ssDNA fragments. These data justify the proposal that glomerular deposition of histones can induce immune complex formation, start an inflammatory process, and produce tissue damage.

The properties of a simple model for solvation in mixed solvents are explored in this paper. The model is based on the supposition that solvent replacement is a simple one-for-one substitution reaction at macromolecular sites which are independent of one another. This leads to a new form for the binding polynomial in which all terms are associated with ligand interchange rather than ligand addition. The principal solvent acts as one of the ligands. Thermodynamic analysis then shows that thermodynamic binding (i.e., selective interaction) depends on the properties of K'-1, whereas stoichiometric binding (site occupation) depends on K'. K' is a 'practical' interchange equilibrium constant given by (f3/f1)K, where K is the true equilibrium constant for the interchange of components 3 and 1 on the site and f3 and f4 denote their respective activity coefficients on the mole fraction scale. Values of K' less than unity lead to negative selective interaction. It is selective interaction and not occupation number which determines the thermodynamic effects of solvation. When K' greater than 100 on the mole fraction scale or K' greater than 2 on the molality scale (in water), the differences between stoichiometric binding and selective interaction become less than 1%. The theory of this paper is therefore necessary only for very weak binding constants. When K'-1 is small, large concentrations of the added solvent component are required to produce a thermodynamic effect. Under these circumstances the isotherms for the selective interaction and for the excess (or transfer) free energy are strongly dependent on the behavior of the activity coefficients of both solvent components. Two classes of behavior are described depending on whether the components display positive or negative deviations from Raoult's law. Examples which are discussed are aqueous solutions of urea and guanidinium chloride for positive deviations and of sucrose and glucose for negative deviations. Examination of the few studies which have been reported in the literature shows that most of the qualitative features of the stabilization of proteins by sugars and their destabilization by urea and guanidinium chloride are faithfully represented with the model. This includes maxima in the free energy of stabilization and destabilization, decreased and zero selective interaction at high concentrations, etc. These phenomena had no prior explanation. Deficiencies in the model as a representation of solvation in aqueous solution are discussed in the appendix.

Flk2/Flt3 is a recently identified receptor tyrosine kinase expressed in brain, placenta, testis, and primitive hematopoietic cells. The mitogenic signalling potential and biochemical properties of Flk2/Flt3 have been analyzed by using a chimeric receptor composed of the extracellular domain of the human colony-stimulating factor 1 receptor and the transmembrane and cytoplasmic domains of murine Flk2/Flt3. We demonstrate that colony-stimulating factor 1 stimulation of the Flk2/Flt3 kinase in transfected NIH 3T3 fibroblasts leads to a transformed phenotype and generates a full proliferative response in the absence of other growth factors. In transfected interleukin 3 (IL-3)-dependent Ba/F3 lymphoid cells, activation of the chimeric receptor can abrogate IL-3 requirement and sustain long-term proliferation. We show that phospholipase C-gamma 1, Ras GTPase-activating protein, the p85 subunit of phosphatidylinositol 3'-kinase, Shc, Grb2, Vav, Fyn, and Src are components of the Flk2/Flt3 signal transduction pathway. In addition, we demonstrate that phospholipase C-gamma 1, the p85 subunit of phosphatidylinositol 3'-kinase, Shc, Grb2, and Src family tyrosine kinases, but not Ras GTPase-activating protein, Vav, or Nck, physically associate with the Flk2/Flt3 cytoplasmic domain. Cell-type-specific differences in tyrosine phosphorylation of p85 and Shc are observed. A comparative analysis of the Flk2/Flt3 signal cascade with those of the endogenous platelet-derived growth factor and IL-3 receptors indicates that Flk2/Flt3 displays specific substrate preferences. Furthermore, tyrosine phosphorylation of p85 and Shc is similarly affected by totally different growth factors in the same cellular background.

Nonalcoholic fatty liver disease (NAFLD) has become a major public health issue. The goal of this study was to assess the clinical use of liver stiffness measurement (LSM) evaluated by supersonic shear imaging (SSI), FibroScan, and acoustic radiation force impulse (ARFI) in a cohort of NAFLD patients who underwent liver biopsy. A total of 291 NAFLD patients were prospectively enrolled from November 2011 to February 2015 at 2 French university hospitals. LSM was assessed by SSI, FibroScan (M probe), and ARFI within two weeks prior to liver biopsy. Calculations of the area under the receiver operating curve (AUROC) were performed and compared for the staging of liver fibrosis. AUROC for SSI, FibroScan, and ARFI were 0.86, 0.82, and 0.77 for diagnoses of ≥F2; 0.89, 0.86, and 0.84 for ≥F3; and 0.88, 0.87, and 0.84 for F4, respectively. SSI had a higher accuracy than ARFI for diagnoses of significant fibrosis (≥F2) (P = 0.004). Clinical factors related to obesity such as body mass index ≥ 30 kg/m(2) , waist circumference ≥102 cm or increased parietal wall thickness were associated with LSM failures when using SSI or FibroScan and with unreliable results when using ARFI. In univariate analysis, FibroScan values were slightly correlated with NAFLD activity score and steatosis (R = 0.28 and 0.22, respectively), whereas SSI and ARFI were not; however, these components of NAFLD did not affect LSM results in multivariate analysis. The cutoff values for SSI and FibroScan for staging fibrosis with a sensitivity ≥90% were very close: 6.3/6.2 kPa for ≥F2, 8.3/8.2 kPa for ≥F3, and 10.5/9.5 kPa for F4.

Although obesity is associated with an increase in LSM failure, the studied techniques and especially SSI provide high value for the diagnosis of liver fibrosis in NAFLD patients. (Hepatology 2016;63:1817-1827).

OBJECTIVE

The ability of an endocrine disruptor exposure during gonadal sex determination to promote a transgenerational prostate disease phenotype was investigated in the current study.

METHODS

Exposure of an F0 gestating female rat to the endocrine disruptor vinclozolin during F1 embryo gonadal sex determination promoted a transgenerational adult onset prostate disease phenotype. The prostate disease phenotype and physiological parameters were determined for males from F1 to F4 generations and the prostate transcriptome was assessed in the F3 generation.

RESULTS

Although the prostate in prepubertal animals develops normally, abnormalities involving epithelial cell atrophy, glandular dysgenesis, prostatitis, and hyperplasia of the ventral prostate develop in older animals. The ventral prostate phenotype was transmitted for four generations (F1-F4). Analysis of the ventral prostate transcriptome demonstrated 954 genes had significantly altered expression between control and vinclozolin F3 generation animals. Analysis of isolated ventral prostate epithelial cells identified 259 genes with significantly altered expression between control and vinclozolin F3 generation animals. Characterization of regulated genes demonstrated several cellular pathways were influenced, including calcium and WNT. A number of genes identified have been shown to be associated with prostate disease and cancer, including beta-microseminoprotein (Msp) and tumor necrosis factor receptor superfamily 6 (Fadd).

CONCLUSIONS

The ability of an endocrine disruptor to promote transgenerational prostate abnormalities appears to involve an epigenetic transgenerational alteration in the prostate transcriptome and male germ-line. Potential epigenetic transgenerational alteration of prostate gene expression by environmental compounds may be important to consider in the etiology of adult onset prostate disease.

Chronic myeloid leukemia (CML) is a stem cell disease in which BCR/ABL promotes the survival of leukemic cells. Heme oxygenase-1 (HO-1) is an inducible stress protein that catalyzes the degradation of heme and has recently been implicated in the regulation of growth and survival of various neoplastic cells. In the present study, we analyzed the expression and role of HO-1 in CML cells. As assessed by Northern and Western blot analysis as well as immunostaining, primary CML cells were found to express HO-1 mRNA and the HO-1 protein in a constitutive manner. Exposure of these cells to the BCR/ABL tyrosine kinase inhibitor STI571 resulted in decreased expression of HO-1 mRNA and protein. In addition, BCR/ABL was found to up-regulate HO-1 promoter activity, mRNA levels, and protein levels in Ba/F3 cells. To investigate the role of HO-1 for survival of primary CML cells, the HO-1 inducer hemin was used. Hemin-induced expression of HO-1 was found to protect CML cells from STI571-induced cell death. In addition, inhibition of HO-1 by zinc-(II)-deuteroporphyrin-IX-2,4-bisethyleneglycol resulted in a substantial decrease of cell viability. Furthermore, overexpression of HO-1 in the CML-derived cell line K562 was found to counteract STI571-induced apoptosis. Together, our data identify HO-1 as a novel BCR/ABL-driven survival molecule and potential target in leukemic cells in patients with CML. The pathogenetic and clinical implications of this observation remain to be elucidated.

The brain topography of power spectra along the antero-posterior (A-P) axis was studied in the all-night human sleep EEG. Spectra (0.25 - 25.0 Hz) were computed for an anterior (A; F3-C3), a middle (M; C3-P3) and a posterior (P; P3-O1) bipolar derivation, and the spectral gradients between two adjacent derivations were expressed by power ratios (A/M and M/P). At NREM-REM sleep transitions a power shift from A to M was present over almost the entire frequency range, while the direction of shifts between M and P differed between frequency bands. Within NREM sleep, frequency specific power gradients were present: In the low delta band power in both A (0.25 Hz bin) and P (0.25-1.0 Hz bins) was higher than in M. In the 4-9 Hz range the relation was A>> M>> P, and in the 15 - 25 Hz range power was largest in M. Power in the spindle frequency range was highest at 11.75 Hz in M, and at 13.5 - 13.75 Hz in A. Topographical differences were seen also in the temporal changes of power across and within NREM sleep episodes. Whereas NREM sleep power in the 2-Hz bin was higher in A than in M in the first episode, this difference vanished in the course of the night. This result points to a specific involvement of frontal parts of the cortex in sleep homeostasis. The regional differences in sleep EEG spectra indicate that sleep is not only a global phenomenon but also a local brain process with a different regional involvement of neuronal populations.

The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit-SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3-c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling of c-Kit engagement to proliferation.

BACKGROUND

Dynamic action of anesthetic agents was compared at cortical and subcortical levels during induction of anesthesia. Unconsciousness involved the cortical brain but suppression of movement in response to noxious stimuli was mediated through subcortical structures.

METHODS

Twenty-five patients with Parkinson disease, previously implanted with a deep-brain stimulation electrode, were enrolled during the implantation of the definitive pulse generator. During induction of anesthesia with propofol (n = 13) or sevoflurane (n = 12) alone, cortical (EEG) and subcortical (ESCoG) electrogenesis were obtained, respectively, from a frontal montage (F3-C3) and through the deep-brain electrode (p0-p3). In EEG and ESCoG spectral analysis, spectral edge (90%) frequency, median power frequency, and nonlinear analysis dimensional activation calculations were determined.

RESULTS

Sevoflurane and propofol decreased EEG and ESCoG activity in a dose-related fashion. EEG values decreased dramatically at loss of consciousness, whereas there was little change in ESCoG values. Quantitative parameters derived from EEG but not from ESCoG were able to predict consciousness versus unconsciousness. Conversely, quantitative parameters derived from ESCoG but not from EEG were able to predict movement in response to laryngoscopy.

CONCLUSIONS

These data suggest that in humans, unconsciousness mainly involves the cortical brain, but that suppression of movement in response to noxious stimuli is mediated through the effect of anesthetic agents on subcortical structures.

Voltage-gated sodium channels isolated from mammalian brain are composed of alpha, beta1, and beta2 subunits. The alpha subunit forms the ion conducting pore of the channel, whereas the beta1 and beta2 subunits modulate channel function, as well as channel plasma membrane expression levels. beta1 and beta2 each contain a single, extracellular Ig-like domain with structural similarity to the neural cell adhesion molecule (CAM), myelin Po. beta2 contains strong amino acid homology to the third Ig domain and to the juxtamembrane region of F3/contactin. Many CAMs of the Ig superfamily have been shown to interact with extracellular matrix molecules. We hypothesized that beta2 may interact with tenascin-R (TN-R), an extracellular matrix molecule that is secreted by oligodendrocytes during myelination and that binds F3-contactin. We show here that cells expressing sodium channel beta1 or beta2 subunits are functionally modulated by TN-R. Transfected cells stably expressing beta1 or beta2 subunits initially recognized and then were repelled from TN-R substrates. The cysteine-rich amino-terminal domain of TN-R expressed as a recombinant peptide, termed EGF-L, appears to be responsible for the repellent effect on beta subunit-expressing cells. The epidermal growth factor-like repeats and fibronectin-like repeats 6-8 are most effective in the initial adhesion of beta subunit-expressing cells. Application of EGF-L to alphaIIAbeta1beta2 channels expressed in Xenopus oocytes potentiated expressed sodium currents without significantly altering current time course or the voltage dependence of current activation or inactivation. Thus, sodium channel beta subunits appear to function as CAMs, and TN-R may be an important regulator of sodium channel localization and function in neurons.

Activation of the natriuretic peptide system lowers blood pressure and causes the excretion of salt. Atrial natriuretic peptide and B-type natriuretic peptide are the humoral mediators of this effect; they act primarily by binding to membrane-bound natriuretic peptide receptor A (NPRA) and stimulating its intrinsic guanylate cyclase activity. To study whether genetically determined differences in NPRA expression affect blood pressure we have generated mice with one, two, three, or four copies of the gene encoding NPRA (Npr1 in the mouse). Atrial natriuretic peptide-dependent guanylate cyclase activity ranged progressively from approximately one-half normal in one-copy animals to twice normal in four-copy animals (P < 0.001). On different diets (0.05%, 2%, and 8% NaCl), the blood pressures of F1 male mice having only one copy of Npr1 averaged 9.1 mmHg (1 mmHg = 133 Pa) above those of wild-type two-copy males (P < 0.001), whereas males with three copies of the gene had blood pressures averaging 5.2 mmHg below normal (P < 0.01). The blood pressures of the one-copy F1 animals were significantly higher (by 6.2 mmHg; P < 0.01) on the high-salt than on the low-salt diet. The blood pressures of four-copy F3 males were significantly lower (by 7 mmHg; P < 0.05) on the high-salt than on the low-salt diet. These results demonstrate that below normal Npr1 expression leads to a salt-sensitive increase in blood pressure, whereas above normal Npr1 expression lowers blood pressures and protects against high dietary salt.