Growth factor-dependent activation of the MAPK pathway in human pancreatic cancer: MEK/ERK and p38 MAP kinase interaction in uPA synthesis

Increased expression of the hepatocyte growth factor (HGF) receptor (c-met) and urokinase type plasminogen (uPA) correlated with the development and metastasis of cancers. To investigate the role of HGF/c-met signaling on metastasis in cancer cells stimulated with HGF, we examined the effects of a specific MEK1 inhibitor (PD98059) and a p38 MAP kinase inhibitor (SB203580) on HGF-induced uPA expression in pancreatic cancer cell lines, L3.6PL and IMIM-PC2. Pretreatment of PD98059 decreased HGF-mediated phosphorylation of extracellular receptor kinase (ERK), uPA secretion and expression of matrix metalloproteinases (MMP-2 and MMP-9) in a dose-dependent manner. In contrast, SB203580 pretreatment increased HGF-stimulated ERK phosphorylation, uPA secretion and expression of MMPs. SB203580 also reversed the inhibition of HGF-mediated ERK activation and uPA secretion in the PD98059-pretreated cells. These results suggest that ERK activation by HGF might play important roles in the metastasis of pancreatic cancer and the p38 MAPK pathway also involved in the HGF-mediated uPA secretion and metastasis by regulation of ERK pathway. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of monoclonal antibodies developed against Sarcocystis neurona

Equine protozoal myeloencephalitis (EPM), caused by a protozoal parasite infection of the central nervous system, is the most commonly diagnosed neurologic disease of horses in North America. In specific regions of the United States approximately 50% of the horse population is seropositive to Sarcocystis neurona. However, not all seropositive horses develop clinical signs. Detailed clinical examination, along with cerebrospinal fluid antibody evaluation are often used to diagnose EPM. Postmortem evaluation of the brain stem and spinal cord for histopathologic lesions compatible with nonsuppurative meningoencephalomyelitis is used for reaching a diagnosis since organisms are difficult to detect by routine staining methods. Immunohistochemical staining aids detection of organisms; however, the polyclonal antibodies that react with S. neurona may react with merozoites of other closely related Sarcocystis species. In this study, two different monoclonal antibodies, mAb 2A7-18 and mAb 2G5-2, were developed against the merozoite stage of S. neurona UCD-SN1 strain. The antibodies were evaluated by immunoblot, immunofluorescence, immuno-electron microscopy and immunohistochemistry for their ability to react with S. neurona. MAb 2G5-2 reacted with antigenically distinct S. neurona isolates whereas mAb 2A7-18 appeared to be limited in its ability to recognize different isolates. These two monoclonal antibodies recognize protein epitopes of two different immunodominant proteins of S. neurona.     

The Validity of the Neale and Kendler Model-Fitting Approach in Examining the Etiology of Comorbidity

Given that knowledge regarding the etiology of comorbidity between disorders can have a significant impact on research regarding the classification, treatment, and etiology of the disorders, the ability to reject incorrect hypotheses regarding the causes of comorbidity is very important. A simulation study was conducted to assess the validity of the Neale and Kendler (1995) model-fitting approach in examining the etiology of comorbidity between two disorders. First, data were simulated under the assumptions of the 13 alternative comorbidity models described by Neale and Kendler. Second, model-fitting analyses testing the comorbidity models were conducted on the simulated datasets. Thirteen sets of data with varying model parameters were simulated to test Neale and Kendler's assertion that their model-fitting approach is appropriate across a range of potential prevalences and degrees of familiality. The validity of the model-fitting approach in examining unselected twin data and a combination of selected family data and unselected family data was explored. The model-fitting approach successfully discriminated several classes of comorbidity models, although discrimination between models within classes of related models was less accurate. Results suggest that the model-fitting approach can be a useful tool in examining the etiology of the comorbidity between disorders if the caveats of the present study's results are considered carefully. As predicted by Neale and Kendler, variations in the disorder prevalences and familial correlations did not affect the validity of their model-fitting approach, but affected the power to discriminate the correct model. As suggested by Neale and Kendler, the model-fitting approach can be applied to both unselected and selected data and to both twin and family data.     

Detection of YMDD motif mutants by oligonucleotide chips in lamivudine-untreated patients with chronic hepatitis B virus infection

Lamivudine, a nucleoside analogue, has been used widely as an effective antiviral agent for the treatment of patients with chronic hepatitis B virus (HBV) infection. However, the YMDD motif mutation of HBV polymerase resistant to lamivudine occurs very frequently after long term therapy. We developed an oligonucleotide chip for the detection of YMDD motif mutants resistant to lamivudine and investigated the prevalence of the mutants in patients with chronic HBV infection who had not been treated by lamivudine before. Forty patients who had not been treated with lamivudine were included in this study. Serum samples were tested by the oligonucleotide chips designed for detection of wild-type YMDD motif, M552V and M552I. Samples were confirmed by restriction fragment length polymorphism (RFLP) and direct sequencing. M552I mutants were detected by the oligonucleotide chips in 7.5% (3/40) of chronic HBV infected patients (2 chronic hepatitis and 1 cirrhosis). The results were in accordance with those of RFLP. YMDD motif mutants occur as natural genome variabilities in patients with chronic HBV infection who had not been treated with lamivudine before. Oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of mutants resistant to antiviral therapy in chronic HBV infection.     

A distinctive interaction between memory and chronic daytime somnolence in asymptomatic APOE e4 homozygotes

STUDY OBJECTIVES: To correlate memory measures with a trait measure of chronic daytime somnolence in cognitively normal individuals with different gene doses of the apolipoprotein E (APOE) e4 allele, a common Alzheimer's disease susceptibility gene. DESIGN: Cross-sectional, exploratory study of cognitive abilities in APOE e4 homozygotes (HMZ) (n=42), heterozygotes (HTZ), (n=42) and noncarriers (NC) (n=42) who are matched for age, gender, educational level, and family history of dementia. SETTING: Tertiary care academic medical center. PARTICIPANTS: Cognitively normal residents of Maricopa County, Arizona who are 30-70 years of age, genotyped for APOE, and have no history of a sleep disorder INTERVENTIONS: N/A. MEASUREMENTS: Epworth Sleepiness Scale (ESS) and a battery of neuropsychological tests RESULTS: Age, education, gender, and insomnia complaints did not significantly differ among groups. Despite normal baseline memory scores, memory declined with increasing ESS on all eight memory measures in the HMZ, two of eight in the HTZ, and one of eight in the NC. Differences between HMZ and NC on the slope of memory decline with increasing ESS reached statistical significance on two verbal memory measures, AVLT Long-Term Memory (p=0.048) and Percent Delayed Recall (p=0.035). CONCLUSIONS: Chronic daytime somnolence is associated with a distinctive decline in verbal memory in cognitively normal APOE e4 HMZ, a group at particularly high risk of Alzheimer's disease. Additional studies are needed to confirm these exploratory findings and to determine the effects of acute somnolence on cognition in these genetic subgroups.     

Glial cell and fibroblast cytotoxicity study on 4026-cyclotene photosensitive benzocyclobutene (BCB) polymer films

Photosensitive benzocyclobutene (photo-BCB) is a class of polymers with the trade name Cyclotene. The photoimagable property of Cyclotene makes it suitable for the manufacture of microelectronic devices. The motivation behind this study is that we see an exciting application of photo-BCB as substrates in implantable microelectronic biomedical devices due to several desirable properties distinctive from other polymer materials. To our knowledge, however, photo-BCB has never been tested for biomedical implant applications, as evidenced by the lack reported data on its biocompatibility. This study takes the first step towards assessing photo-BCB biocompatibility by evaluating the cytotoxicity and cell adhesion behavior of Cyclotene 4026 coatings exposed to monolayers of glial and fibroblast cells in vitro. It can be concluded from these studies that photo-BCB films deposited on silicon wafers using microfabrication processes did not adversely affect 3T3 fibroblast and T98-G glial cell function in vitro. We also successfully rendered photo-BCB films non-adhesive (no significant fibroblast or glial cell adhesion) with surface immobilized dextran using methods developed for other biomaterials and applications. Future work will further develop prototype photo-BCB microelectrode devices for chronic neural implant applications.     

A Novel Mutation in the GATA1 Gene Associated with Acute Megakaryoblastic Leukemia in a Korean Down Syndrome Patient

Although acquired mutations in the GATA1 gene have been reported for Down syndrome-related acute megakaryoblastic leukemia (DS-AMKL) in Caucasians, this is the first report of a Korean Down syndrome patient with AMKL carrying a novel mutation of the GATA1 gene. A 3-yr-old Korean girl with Down syndrome was admitted to our hospital complaining of pallor and fever. The findings of a peripheral blood smear and bone marrow study were compatible with the presence of AMKL. A chromosome study showed 48,XX,-7,+21c,+21,+r[3]/47,XX,+21c[17]. Following GATA1 gene mutation analysis, a novel mutation, c.145dupG (p.Ala49GlyfsX18), was identified in the N-terminal activation domain of the GATA1 gene. This mutation caused a premature termination at codon 67 and expression of an abnormal GATA-1 protein with a defective N-terminal activation domain, and the absence of full-length GATA-1 protein. This case demonstrates that a leukemogenic mechanism for DS-AMKL is contributed by a unique collaboration between overexpressed genes from trisomy 21 and an acquired GATA1 mutation previously seen in Caucasians and now in a Korean patient.     

Fibroblast growth factor 2 induces differentiation and apoptosis of Askin tumour cells

Peripheral primitive neuroectodermal tumour (PNET)/Ewing's sarcoma (ES) and neuroblastoma (NB) are related tumours of neural crest origin with primitive neural characteristics. Fibroblast growth factor 2 (FGF2) is a critical signalling molecule for primitive neural crest cells. The treatment of NB cells with FGF2 variably affects biological characteristics such as growth and differentiation, while in PNET/ES, FGF2 predominantly induces apoptosis. The JK-GMS Askin tumour cell line can be induced to differentiate upon treatment with nerve growth factor (NGF), indicating the integrity of the cellular machinery necessary for differentiation. The present study assesses whether FGF2 can induce differentiation in JK-GMS cells. JK-GMS cells expressed high-affinity FGF receptors (FGFRs), and treatment with FGF2 induced phosphorylation of FGFR1 together with activation of extracellular signal-regulated kinases (ERK1/ERK2) and c-Jun N-terminal kinase (JNK). Subsequent biological effects were growth inhibition, neuronal differentiation, and apoptosis, and these changes were associated with increased expression of neurofilaments, reduction of c-myc and bcl-2 expression, and activation of caspase 3. Treatment of the cells with a specific inhibitor of the MAPK/extracellular signal-regulated kinase (MEK)-1, PD98059, predominantly inhibited the effects of FGF2 on growth, differentiation, and apoptosis, while an inhibitor of JNK reduced apoptosis, indicating that the ERK1/2 and JNK pathways are critical components of FGF2-mediated effects in JK-GMS cells. Additional comparative analyses of FGF2-mediated effects in two ES cell lines (CADO-ES, RD-ES) and a PNET cell line (SK-N-MC) showed pronounced differentiation in SK-N-MC, but not in CADO-ES or RD-ES cells. This study demonstrates that FGF2 can induce neuronal differentiation of PNET including Askin tumour. These findings clearly indicate that the FGF2-mediated signalling pathway plays a critical role in controlling the major properties of PNET cells and may provide a potential therapeutic target for PNET.     

Activation of SiO2-supported zirconocene catalysts by common trialkylaluminiums

A modified SiO₂ was prepared by reacting SiO₂ with Cl₂Si(CH₃)₂ in toluene, on which methylaluminoxane (MAO) was supported to obtain a catalyst precursor. The mixture of the precursor and Cp₂ZrCl₂ (Cp: cyclopentadienyl) gave polyethylene in a high yield even by using common trialkylaluminiums as cocatalyst. Surprisingly, the MAO-free catalyst system composed of the modified SiO₂ and Cp₂ZrCl₂ was also found to be activated by common trialkylaluminiums.     

Loss of fibroblast Thy-1 expression correlates with lung fibrogenesis

Fibroblasts consist of heterogeneous subpopulations that have distinct roles in fibrotic responses. Previously we reported enhanced proliferation in response to fibrogenic growth factors and selective activation of latent transforming growth factor (TGF)-beta in fibroblasts lacking cell surface expression of Thy-1 glycoprotein, suggesting that Thy-1 modulates the fibrogenic potential of fibroblasts. Here we report that compared to controls Thy-1-/- C57BL/6 mice displayed more severe histopathological lung fibrosis, greater accumulation of lung collagen, and increased TGF-beta activation in the lungs 14 days after intratracheal bleomycin. The majority of cells demonstrating TGF-beta activation and myofibroblast differentiation in bleomycin-induced lesions were Thy-1-negative. Histological sections from patients with idiopathic pulmonary fibrosis demonstrated absent Thy-1 staining within fibroblastic foci. Normal lung fibroblasts, in both mice and humans, were predominantly Thy-1-positive. The fibrogenic cytokines interleukin-1 and tumor necrosis factor-alpha induced loss of fibroblast Thy-1 surface expression in vitro, which was associated with Thy-1 shedding, Smad phosphorylation, and myofibroblast differentiation. These results suggest that fibrogenic injury promotes loss of lung fibroblast Thy-1 expression, resulting in enhanced fibrogenesis.