Mutational analysis of Fas ligand gene in human non-Hodgkin lymphoma

Among the systems triggering apoptosis, the Fas-Fas ligand (FasL) system is recognized as a major pathway for the induction of apoptosis in cells and tissues. Ligation of Fas by either an agonistic antibody or FasL transmits a 'death signal' to the target cell, potentially triggering apoptosis. Alterations of genes along the Fas-mediated apoptosis pathway have been reported in many human cancers. However, there have been no data regarding FasL gene mutations in human cancers. We hypothesized that FasL gene mutation might be involved in the development of non-Hodgkin lymphoma (NHL). In this study, we analyzed the entire coding region of the FasL gene for the detection of somatic mutations in a series of 111 NHLs and found that one tumor had a FasL gene mutation in the cytoplasmic domain. To evaluate the functional alterations of the mutant in apoptosis, we overexpressed the mutant in 293T cells, but couldn't find any significant loss of cell death compared to the wild-type FasL. Together, these data suggest that FasL is occasionally mutated in human NHL and that FasL mutations appear to play no role in the pathogenesis of the vast majority of NHLs.     

Comparison of deltaFVC between patients with allergic rhinitis with airway hypersensitivity and patients with mild asthma.

BACKGROUND: In asthmatic individuals, airway sensitivity and maximal airway response are increased. Airway sensitivity is usually evaluated by measuring the provocation concentration of inhaled methacholine or histamine that causes a decrease in forced expiratory volume in 1 second of 20% (PC20). The percentage decrease in forced vital capacity at the PC20 (deltaFVC) has been proposed as a surrogate marker for maximal airway response. Individuals with allergic rhinitis and no clinical evidence of asthma frequently exhibit airway hypersensitivity. OBJECTIVE: To compare the deltaFVC between patients with allergic rhinitis and mild asthmatic patients with a similar degree of airway hypersensitivity. METHODS: A retrospective analysis of methacholine challenge test data from 72 children with allergic rhinitis and airway hypersensitivity (methacholine PC20 < 16 mg/mL) (rhinitis group) and from 72 children with mild atopic asthma matched to the rhinitis group regarding the methacholine PC20 (asthma group). The deltaFVC was calculated on the concentration-response curve to methacholine. RESULTS: The mean +/- SD deltaFVC was significantly lower in the rhinitis group (15.0% +/- 3.6%) vs the asthma group (17.4% +/- 5.3%) (P = .002). There was no significant correlation between the deltaFVC and PC20 in the rhinitis (r = -0.101; P = .41) and asthma (r = -0.023; P = .85) groups when 2 patients with PC20 less than 1 mg/mL were excluded from each group. CONCLUSIONS: Patients with allergic rhinitis and airway hypersensitivity had a significantly lower deltaFVC than methacholine PC20-matched mild asthmatic patients, suggesting that the level of maximal airway response in patients with allergic rhinitis is lower than that in mild asthmatic patients with a similar degree of airway hypersensitivity.     

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.     

Monoacyl lipoteichoic acid from pneumococci stimulates human cells but not mouse cells

We have developed a method for obtaining pneumococcal lipoteichoic acid (LTA) with none, one, or two acyl chains. Anion-exchange chromatography at pH 9.5 yields pneumococcal LTA (labeled LTA-9.5) that has a mass spectrum identical to that of pre-ion-exchange LTA and loses 500 mass units after deacylation by alkali hydrolysis. Anion exchange at pH 10.5 produces LTA (labeled LTA-10.5) with mass peaks that are 264 mass units lower than those of pre-ion-exchange LTA, and deacylation of LTA-10.5 by alkali hydrolysis reduces the mass by only 239 mass units. This result indicates that LTA-10.5 has lost one of the two acyl chains, whereas LTA-9.5 has both acyl chains. When the biological properties of LTA-9.5 and LTA-10.5 are examined with mouse cells, only LTA-9.5 (and not LTA-10.5) is able to stimulate mouse cells to produce tumor necrosis factor alpha, interleukin-1beta, and nitric oxide. In contrast, both LTA-9.5 and LTA-10.5 can stimulate human cells. LTA became inactive when both acyl chains were removed. Thus, acyl chains are critical for LTA function, and small variations in acyl chains can alter biological properties of LTA.     

Norepinephrine transporter (NET) promoter and 5'-UTR polymorphisms: association analysis in panic disorder

Several biochemical and pharmacological studies suggest that the catecholaminergic system involving the norepinephrine transporter (NET) is relevant for the pathogenesis of panic disorder. Three single nucleotide polymorphisms in the promoter or untranslated 5' region of the NET gene were investigated by means of RFLP analysis in a sample of 115 German patients with panic disorder and 115 matched controls. Statistical analysis failed to show association with the overall diagnosis of panic disorder. In the subgroup of patients with panic disorder without agoraphobia, however, two polymorphisms were found to be associated with the disease (G/C (rs2397771): p < 0.05; T/C (rs2242446): p < 0.01). While our data do not support a major function of the NET gene in the development of panic disorder, it may play a role in the subgroup of panic disorder without agoraphobia.     

Loss of heterozygosity on chromosomes 3p,8p,9p and 17p in the progression of squamous cell carcinoma of the larynx

Previous molecular genetic studies of laryngeal squamous cell carcinoma (SCC)have shown certain chromosomal regions with recurring alterations. But studies of sequential molecular alterations and genetic progression model of laryngeal SCC have not been clearly defined. To identify the chromosomal alterations associated with the carcinogenesis of laryngeal SCC, we analyzed genomic DNA from microdissected squamous metaplasia, squamous dysplasia, invasive SCC, and metastatic carcinoma samples from 22 laryngeal SCC patients for loss of heterozygosity (LOH) at microsatellite loci. Ten microsatellite markers on chromosome 3p, 8p, 9p, and 17p were used. LOH at 9p21 was observed in the all stages including squamous metaplasia, squamous dysplasia, invasive SCC and metastatic carcinoma. LOH at 17p13.1, 3p25 and 3p14.2 was observed from the squamous dysplasia, invasive SCC and metastatic carcinoma. LOH at 8p21.3-p22 was observed mainly from the invasive SCC and metastatic carcinoma. The results suggest that 9p21 in the early event, 17p13.1, 3p25 and 3p14.2 in the intermediate event and 8p21.3- p22 in the late event may be involved in the laryngeal carcinogenesis.     

Methods for derivation of human embryonic stem cells

The expanded blastocysts, developed from 2PN-stage embryos, are generally divided into three categories: a good blastocyst containing a large and distinguishable inner cell mass (ICM), a blastocyst with a small and distinct ICM, and a blastocyst with a poorly defined ICM. In this study, we introduce methods for the derivation of human embryonic stem cells (hESCs) depending on the quality of the blastocysts. An immunosurgical method was used for the good expanded blastocysts. This method, however, raises the probability of ICM loss in cases of hESC derivation from blastocysts with smaller or indistinct ICMs. Furthermore, this method is also associated with a risk of the contamination of the hESCs with animal pathogens. To overcome these shortcomings, the partial- or whole-embryo culture method was used. For blastocysts with no visible ICM, the whole-embryo culture method was used to establish hESCs via the seeding of the entire blastocyst without its zona pellucida directly on a STO feeder layer. However, trophectodermal overgrowth tends to hinder the expansion of the ICM during the initial steps of hESC derivation. Therefore, the partial-embryo culture method was developed to establish hESCs from blastocysts with smaller ICMs. The surgical isolation of the region containing the ICM with an ultra-fine glass pipette alleviates trophectoderm overgrowth. This method is also applicable to blastocysts with large and distinct ICMs, and the efficiency of this method is comparable to that of the immunosurgical method.