A genetic mouse model carrying the nonfunctional xeroderma pigmentosum group G gene

ABSTRACT  A genetic mouse model with a disrupted XPG allele was generated by insertion of neo cassette sequences into exon 3 of the XPG gene by using embryonic stem (ES) cell techniques. The xpg -deficient mice showed distinct developmental characteristics. Their body was marked smaller than that in wild-type littermates since the postnatal day 6, and this postnatal growth failure became more severe with developmental proceeding. Their life span was very short, all of the mutants died by postnatal day 23 after showing great weakness and emaciation. In addition, the mutant homozygous mice also showed some progressive neurological signs, like the lower level of activity and a progressive ataxia. Further examination indicated there was developmental retardation of the brain in the mutant mice. Their brain weight, and thickness of cerebral cortex and cerebellar cortex were significant different from the controls. These characteristics, like small size brain, brain developmental retardation and progressive neurological dysfunctions in the homozygotes were similar to the typical clinical phenotype of the XPG patients and Cockayne syndrome, we believe that the xpg-deficient mice will be an animal model for studying the function of the XP-G protein in nucleotide-excision repair and mechanisms related to the clinic symptoms of XP-G and Cockayne syndrome in humans.     

The effects of stimulating protease-activated receptor-1 and -2 in A172 human glioblastoma

Summary. Human glioblastoma cell line A172 expressed protease-activated receptor-1 and -2 (PAR-1 and PAR-2). We investigated the effects of the stimulation of these receptors by receptor-activating agonist peptides on the Ca²⁺ signaling, protein kinase C translocation, cell morphology and cell proliferation in A172. Both PAR-1 agonist SFLLRN and PAR-2 agonist SLIGKV induced an increase in [Ca²⁺]i. The prior treatment of A172 with PAR-2 agonist SLIGKV did not influence the [Ca²⁺]i response to PAR-1 agonist SFLLRN or thrombin, however, the prior treatment with PAR-1 agonist SFLLRN or thrombin completely abolished the second response to PAR-2 agonist SLIGKV. Treatment with each agonist peptide produced thinner and fewer processes in A172. The PAR-2 agonist inhibited the proliferation of A172 significantly while PAR-1 agonist did not. PKC-α and γ were translocated from cytosol to membrane with either PAR-1 or PAR-2 stimulation, however, ι was specifically translocated with SFLLRN, and λ with SLIGKV, respectively. These results indicated that PAR-1 and PAR-2 stimulation produced a similar [Ca²⁺]i response and morphological changes in A172 glioblastoma while the effects on the cell proliferation and activation of PKC isozymes were distinct, suggesting that different signal transduction pathways were activated by these receptors. The uni-directional cross desensitization implies a functional linkage between PAR-1 and PAR-2 receptors. Received May 23, 2000; accepted September 13, 2000     

Genetic perspectives on the serotonin transporter

The serotonin transporter (5-HTT) is most well known as the site of action of the serotonin reuptake inhibitors, which were initially developed as antidepressants, but now are the most widely used agents in the treatment of many additional neuropsychiatric and related disorders. The discovery that the gene that expresses the 5-HTT possesses a functional promoter-region polymorphism, which is associated with temperament and personality traits such as anxiety and negative emotionality as well as some behaviors, led to many studies examining this polymorphism in individuals with different neuropsychiatric disorders. The subsequent development of mice with a targeted disruption of the 5-HTT in our laboratory has provided an experimental model to examine the many consequences of diminished (in +/-, heterozygote mice) or absent (in -/-, homozygote knockout mice) function of the 5-HTT. The 5-HTT-deficient mice were also crossed with other knockout mice, allowing the study of multiple neurobiologic dysfunctions. As multiple genes are probably involved in the expression of complex behaviors such as anxiety, as well as neuropsychiatric disorders, these more genetically complex mice may more closely model disorders with complex etiologies. Thus, the combination of these comparative human and mouse studies may extend the opportunities to examine genetic alterations from a novel "bottom-up" approach [gene knockout or partial gene knockout in a combinational gene x gene x (yet unknown) gene approach], which is complementary to the traditional "top-down" genetic approach based upon studies of individuals with diagnosed neuropsychiatric disorders and their family members.     

Bcl-2 mediated inhibition of erucylphosphocholine-induced apoptosis depends on its subcellular localisation

The synthetic phospholipid derivative erucylphosphocholine (ErPC) is a potent inducer of apoptosis in human tumor cell lines. This membrane-targeted drug induces apoptosis independently from death receptor signaling through a mitochondrial pathway that is inhibited by over-expression of Bcl-2.Within the cell, Bcl-2 resides in membranes of mitochondria, endoplasmic reticulum (ER) and the nucleus. However, the importance of its subcellular localisation in distinct organelles for protection against apoptosis is not completely understood.To investigate the impact of Bcl-2 localised at defined subcellular compartments on its protective effects against ErPC-induced apoptosis, Bcl-2 expression was directed to the outer membrane of the mitochondria or the ER of Jurkat T Lymphoma cells, using Bcl-2 mutants with modified membrane anchors. The mitochondrial insertion sequence of ActA directed Bcl-2 to the mitochondria (Bcl-2/MT), the ER-specific sequence of cytochrome b5 to the ER (Bcl-2/ER). Additionally, Jurkat cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/ΔTM) were employed.While restricted expression of Bcl-2 either at membranes of the mitochondria or the ER strongly interfered with ErPC-induced mitochondrial damage and apoptosis, cytosolic Bcl-2/ΔTM exhibited only reduced protection. Thus, membrane localisation of Bcl-2 is a prerequisite for substantial protection against ErPC-induced apoptosis. For efficient long-term inhibition of ErPC-induced apoptosis Bcl-2 had to be present in the membranes of both compartments, the ER and the mitochondria.The finding that ER-targeted Bcl-2 interferes with ErPC-induced mitochondrial damage points to an involvement of the ER in apoptosis signaling upstream of the mitochondria and to a crosstalk between both compartments.     

Discovery of Aminopyrazole Derivatives as Potent Inhibitors of Wild-Type and Gatekeeper Mutant FGFR2 and 3

Fibroblast growth factor receptors (FGFR) 2 and 3 have been established as drivers of numerous types of cancer with multiple drugs approved or entering late stage clinical trials. A limitation of current inhibitors is vulnerability to gatekeeper resistance mutations. Using a combination of targeted high-throughput screening and structure-based drug design, we have developed a series of aminopyrazole based FGFR inhibitors that covalently target a cysteine residue on the P-loop of the kinase. The inhibitors show excellent activity against the wild-type and gatekeeper mutant versions of the enzymes. Further optimization using SAR analysis and structure-based drug design led to analogues with improved potency and drug metabolism and pharmacokinetics properties.     

闽东地区畲族人群CYP2C19基因多态性分析

目的：调查闽东地区汉族与畲族人群CYP2C19基因多态性分布情况。方法：采用限制性片段长度多态性聚合酶链反应（PCR-RFLP）,对汉族（n=140）与畲族（n=180）人群CYP2C19两个多态性位点CYP2C19·2和 CYP2C19·3的基因多态性进行分析,用SPSS16.0软件进行统计分析,Hardy-Weinberg遗传平衡定律验证基因型分布情况。结果：汉族与畲族人群各种基因型出现频率分别为：·1/·1 14.3%,28.9%;·1/·2 28.6%,30.0%;·1/·3 11.4%,16.7%;·2/·2 21.4%,9.4%;·2/·3 21.4%,12.8%;·3/·3 2.9%,2.2%。汉族与畲族CYP2C19不同等位基因出现频率：·1为34.3%,52.2%;·2为46.4%,30.8%;·3为19.3%,16.9%。结论：闽东地区畲族与汉族CYP2C19·2与·3等位基因突变率存在差异,畲族人群基因突变率低于汉族（P＜0.05）。     

Inhalation delivery dramatically improves the efficacy of topotecan for the treatment of local and distant lung cancer

Topotecan is potent anti-cancer drug approved for various malignancies but hematopoietic toxicities undermine its wider application and use of its most effective dose. This study aims to improve these limitations through inhalation-delivery. The pharmacokinetics, efficacy, and toxicity of 2–5 times lower inhalation doses of topotecan dry-powder were compared with the standard intravenous (IV) delivery once/twice-a-week. Human-derived EGFR-mutant (H1975), KRAS-mutant (A549), and EGFR/KRAS wild-type (H358) orthotopic and distant lung tumors were evaluated in murine models. Inhalation of 1 mg/kg topotecan significantly improved the half-life and drug exposure (area under the curve, AUC) compared to 5 mg/kg via IV-delivery. AUCs (h*ng/mL) for inhaled/IV topotecan in plasma, lung, liver, and brain were, 831/888, 60,000/1080, 8380/4000, and 297/15, respectively; while the half-life was also greatly increased in these tissues. The average lung tumor burden of H358-derived tumors was reduced from 15.0 g to 8.4 g (44%) in rats treated once-a-week with 2 mg/kg IV and 1.8 g (88%) with 1 mg/kg inhaled topotecan, corroborating previous findings using A549- and H1975-derived orthotopic lung tumors. Importantly, inhaled topotecan showed superior efficacy in suppressing lung tumors at distant sites. The growth of H1975- and H358-derived subcutaneous xenografts were completely arrested and A549-derived tumors were significantly reduced in mice treated twice-a-week with 1 mg/kg inhaled topotecan compared to a minor (H1975 and H358) or no reduction (A549) with twice-a-week 5 mg/kg IV topotecan.     

mCPP-induced hyperactivity in 5-HT2C receptor mutant mice is mediated by activation of multiple 5-HT receptor subtypes

The serotonin receptor agonist mCPP induces hyperlocomotion in 5-HT2C receptor knockout (KO) mice or in the presence of a 5-HT2C receptor antagonist. In the present group of experiments, we evaluate the role of 5-HT1A, 5-HT1B and 5-HT2A receptors in mCPP-induced hyperactivity in 5-HT2C KO mice. We also assess the ability of agonists at these receptors to induce hyperactivity in wildtype (WT) mice pre-treated with a selective 5-HT2C receptor antagonist. As previously reported, mCPP (3 mg/kg) induced hyperactivity in 5-HT2C KO mice. A combination of the 5-HT1B receptor agonist CP-94,253 (20 mg/kg) and the 5-HT1A receptor agonist 8-OH-DPAT (0.5 mg/kg) induced marked hyperactivity in WT but not in 5-HT2C KO mice, nor in mice treated with the selective 5-HT2C receptor antagonist, SB 242084 (1.5 mg/kg). Neither CP-94,253 nor 8-OH-DPAT had any intrinsic effect on locomotion in WTs. mCPP-induced hyperactivity was attenuated in 5-HT2C KO mice by the 5-HT1B receptor antagonist SB 224289 (2.5 mg/kg), and the 5-HT2A receptor antagonists ketanserin (0.3 mg/kg) and M100907 (0.01 mg/kg) but not by the 5-HT1A receptor antagonist WAY 100635 (1 mg/kg). The 5-HT(2A/2B/2C) receptor agonist, Ro 60-0175 (3 mg/kg), induced a modest increase in locomotor activity in WT mice pre-treated with SB 242084. However, the combination of Ro 60-0175 with CP-94,253 induced a substantial increase in activity in 5-HT2C KO mice, an effect comparable to mCPP-induced hyperactivity. Thus, joint activation of 5-HT1A and 5-HT1B receptors stimulates locomotion in WT mice but this response is dependent on a functional 5-HT2C receptor population and hence is absent in 5-HT2C KO mice. By contrast, mCPP-induced hyperactivity depends on the inactivation of a separate 5-HT2C receptor population and is mediated by 5-HT2A and 5-HT1B receptor activation.     

Identification of a zebrafish model of muscular dystrophy

1. Large-scale mutagenic screens of the zebrafish genome have identified a number of different classes of mutations that disrupt skeletal muscle formation. Of particular interest and relevance to human health is a class of recessive lethal mutations in which muscle differentiation occurs normally, but is followed by tissue-specific degeneration reminiscent of human muscular dystrophies. 2. We have shown that one member of this class of mutations, sapje (sap), results from mutations within the zebrafish orthologue of the human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology by disrupting the link between the actin cytoskeleton and the extracellular matrix in skeletal muscle cells. 3. We have found that the progressive muscle degeneration phenotype of sapje-mutant zebrafish embryos is caused by the failure of somitic muscle attachments at the embryonic myotendinous junction (MTJ). 4. Although a role for dystrophin at the MTJ has been postulated previously and MTJ structural abnormalities have been identified in the dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure is thus far lacking. Therefore, the sapjre mutation may provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases. In the present review, we discuss this finding in light of previously postulated models of dystrophin function.