Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members

Unique among fibroblast growth factors (FGFs), FGF19, -21, and -23 act in an endocrine fashion to regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis. These FGFs require the presence of Klotho/betaKlotho in their target tissues. Here, we present the crystal structures of FGF19 alone and FGF23 in complex with sucrose octasulfate, a disaccharide chemically related to heparin. The conformation of the heparin-binding region between beta strands 10 and 12 in FGF19 and FGF23 diverges completely from the common conformation adopted by paracrine-acting FGFs. A cleft between this region and the beta1-beta2 loop, the other heparin-binding region, precludes direct interaction between heparin/heparan sulfate and backbone atoms of FGF19/23. This reduces the heparin-binding affinity of these ligands and confers endocrine function. Klotho/betaKlotho have evolved as a compensatory mechanism for the poor ability of heparin/heparan sulfate to promote binding of FGF19, -21, and -23 to their cognate receptors.     

Understanding the molecular basis of Apert syndrome

Apert syndrome, first described in 1906, is one of the most severe of the craniosynostosis syndromes and is further characterized by midface hypoplasia, syndactyly, and other visceral abnormalities. Affected individuals generally require lifelong management by a multidisciplinary team of health care specialists. Apert syndrome results almost exclusively from one or the other of two point mutations in fibroblast growth factor receptor 2. Tremendous scientific advances have been made recently in understanding the molecular basis for Apert syndrome through clinical genetic, biochemical, and structural approaches. In this review, the authors provide the clinician with a basic overview of these findings and their therapeutic implications.     

Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection

Background

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs.

Results

Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20–22 h p.i.), but the logarithmic infection phase (days 2–3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-γ (IFN-γ), as well as the more-potent experimental antiviral agent AK-2.

Conclusion

The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.     

Influence of Heparin Mimetics on Assembly of the FGF·FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM₆), octasaccharide (HM₈), and decasaccharide (HM₁₀), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM₈ and HM₁₀ are significantly more potent than HM₆ in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM₈ relative to HM₆ in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM₈ to bind and dimerize FGF2. Notably FGF2·HM₈ exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.     

Evaluation of the effectiveness of conventional insecticides against the cowpea weevil, Callosobruchus maculatus (Coleoptera: Bruchidae), on four different substrata surfaces

在害虫治理中,在消费或贮藏粮食加工产品的建筑设施或场所进行害虫防治需要将杀虫剂施用在各种基质表面上.为了测定不同基质表面上杀虫剂的药效,将四纹豆象Callosobruchus maculatus(F.)成虫接触田间推荐剂量的阿维菌素、溴氰菊酯和毒死蜱.结果表明:施用在玻璃、瓷砖、塑料和纸盘表面上,阿维菌素对四纹豆象成虫的致死率分别为63.33％,22.41％,12.9％和11.9％,而溴氰菊酯在这4种基质表面上对四纹豆象成虫的致死率分别为55％,44.2％,41.3％和37.4％.在所有基质表面上接触毒死蜱,四纹豆象成虫的死亡率均为100％.对数据进行的Probit分析表明,毒死蜱制剂在玻璃、瓷砖、塑料和纸盘上对四纹豆象成虫的LC50值分别为8.66,13.6,29 16和56.5 μg/mL,阿维菌素制剂的相应数值分别为119.4,446.2,774.2和836.4μg/mL,溴氰菊酯制剂的相应数值分别为1008,1131,1210和1336 μg/mL.据此推断,毒死蜱对四纹豆象的毒性最强,且在玻璃、瓷砖、塑料和纸盘表面上的毒性依次降低.     

Evaluation of physiological importance of metallothionein protein expressed by Tetrahymena cadmium metallothionein 1 (TMCd1) gene in Escherichia coli

TMCd1 is a cadmium inducible metallothionein (MT) gene. In the present study the TMCd1 gene of a ciliate protozoan has been expressed in E. coli and the function of the expressed TMCd1 protein as a metal-binding protein has been evaluated. The growth of E. coli cells expressing the GST fused TMCd1 proteins in the presence of cadmium metal clearly demonstrated the role of TMCd1 as a metal-binding protein. The metal accumulation experiments showed that the bacterial cells expressing the functional TMCd1 protein accumulated 19-fold more cadmium in contrast to control cells that lacked the TMCd1 protein expression. The results clearly demonstrate a physiological role of full length TMCd1 protein of a ciliate, expressed in E. coli, in cadmium metal sequestration and detoxification.     

Identification of candidate SNPs for drug induced toxicity from differentially expressed genes in associated tissues

The growing collection of publicly available high-throughput data provides an invaluable resource for generating preliminary in silico data in support of novel hypotheses. In this study we used a cross-dataset meta-analysis strategy to identify novel candidate genes and genetic variations relevant to paclitaxel/carboplatin-induced myelosuppression and neuropathy. We identified genes affected by drug exposure and present in tissues associated with toxicity. From ten top-ranked genes 42 non-synonymous single nucleotide polymorphisms (SNPs) were identified in silico and genotyped in 94 cancer patients treated with carboplatin/paclitaxel. We observed variations in 11 SNPs, of which seven were present in a sufficient frequency for statistical evaluation. Of these seven SNPs, three were present in ABCA1 and ATM, and showed significant or borderline significant association with either myelosuppression or neuropathy. The strikingly high number of associations between genotype and clinically observed toxicity provides support for our data-driven computations strategy to identify biomarkers for drug toxicity.     

Factors Influencing Realized Sex Ratios in Fig Wasps: Double Oviposition and Larval Mortalities

Pollinator fig wasps (Agaonidae) are a model system for studies of sex ratio evolution. They lay their eggs in galled ovules within figs. Only one adult emerges from each gall, suggesting that only one egg is always laid per ovule, but if double oviposition occurs then the assumption that adult (realised) sex ratios of fig wasps are representative of primary sex ratios may be violated. Many galls also fail to produce any wasps. If they initially contained eggs then differential mortality rates may also modify realized sex ratios. We investigated whether Kradibia (= Liporrhopalum) tentacularis foundresses in Ficus montana figs avoid laying in ovules that already contain eggs. Comparisons of oviposition frequencies and wasp emergence frequencies showed that most galls that failed to produce wasps will have had eggs laid in them, but few occupied ovules contained two eggs. Realised sex ratios therefore do not necessarily reflect primary sex ratios in this species, but double oviposition is not responsible.