The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes.

Glycosylphosphatidylinositol (GPI)-anchored proteins are nonmembrane spanning cell surface proteins that have been demonstrated to be signal transduction molecules. Because these proteins do not extend into the cytoplasm, the mechanism by which cross-linking of these molecules leads to intracellular signal transduction events is obscure. Previous analysis has indicated that these proteins are associated with src family member tyrosine kinases; however, the role this interaction plays in the generation of intracellular signals is not clear. Here we show that GPI-anchored proteins are associated with alpha subunits of heterotrimeric GTP binding proteins (G proteins) in both human and murine lymphocytes. When the GPI-anchored proteins CD59, CD48, and Thy-1 were immunoprecipitated from various cell lines or freshly isolated lymphocytes, all were found to be associated with a 41-kDa phosphoprotein that we have identified, by using specific antisera, as a mixture of tyrosine phosphorylated G protein alpha subunits: a small amount of Gialpha1, and substantial amounts of Gialpha2 and Gialpha3. GTP binding assays performed with immunoprecipitations of CD59 indicated that there was GTP-binding activity associated with this molecule. Thus, we have shown by both immunochemical and functional criteria that GPI-anchored proteins are physically associated with G proteins. These experiments suggest a potential role of G proteins in the transduction of signals generated by GPI-anchored molecules expressed on lymphocytes of both mouse and human.     

The relationship between acute ischaemic stroke and plasma D-dimer levels in patients developing neither venous thromboembolism nor major intercurrent illness.

The true relationship between plasma D-dimers and acute ischaemic stroke (AIS) is uncertain as previous studies investigating this have not screened for subclinical deep vein thrombosis. We addressed this as part of a study in which we screened AIS patients for venous thromboembolism (VTE). We also assessed the performance characteristics of two D-dimer assays as exclusionary tests for VTE in these patients. One hundred and two unselected AIS patients were screened for VTE using magnetic resonance direct thrombus imaging. D-dimers were analysed on days 2, 9, 14 and 21 using the VIDAS immunofluorescent assay (cut-off >or= 500 ng/ml) and the IL test D-dimer immunoturbidimetric assay (cut-off >or= 255 ng/ml). The relationship between D-dimers and AIS was examined in 52 patients neither developing VTE nor intercurrent illness. D-dimers were elevated throughout the study. Median values at the four time points were 652, 692, 737 and 686 ng/ml (VIDAS assay) and 260.5, 268.5, 273 and 283 ng/ml (IL assay). D-dimers were higher in patients aged older than 70 years, with severe stroke or with total anterior circulation infarcts: only age older than 70 years was significantly associated with D-dimer values greater than the median on univariate and multivariable analysis. Both assays were 100% sensitive for VTE. Specificities were 30% (VIDAS assay) and 34% (IL assay). Specificity was adversely affected by age older than 70 and severe versus non-severe stroke. D-dimers are elevated in the first 3 weeks post-AIS after eliminating the confounding effect of subclinical deep vein thrombosis. The VIDAS and IL assays remained sensitive tests for VTE but the specificity was low, limiting their exclusionary efficiency in these patients.     

The analysis of the fibroblast growth factor ligand-receptor complex using a quartz crystal microbalance-dissipation

Introduction   The quartz crystal microbalance-dissipation (QCM-D) is a device that measures both the mass of adsorbates upon a surface and the energy dissipated by the surface. The latter quantity, known as dissipation (D), gives information about the viscoelastic properties of species adsorbed to the surface leading to insights into their shape and conformation.
Methods   The fibroblast growth factor (FGF) receptor-ligand signalling complex is simulated within the QCM-D by the immobilization of heparin-derived oligosaccharides to a gold-sputtered QCM-D crystal surface, and then the surface is interrogated with various growth factors and a recombinant receptor. Fibroblast growth factor receptor 1 (FGFR1) extracellular domain (immunoglobulin loops II and III and the acid box of the FGFR1IIIc), FGF-1, FGF-2, FGF-7 and hepatocyte growth factor/scatter factor (HGF/SF) all bind to immobilized heparin-derived oligosaccharides with different changes in D relative to mass for the same surface.
Results   For example, complexes of FGF-1 and octasaccharide are, on a mass basis, far more dissipative than complexes of HGF/SF. One interpretation of these results is that octasaccharides are relatively flexible, or at least contain hinge regions and that different FGFs have a preference for binding above the hinge (so more dissipative) or on the hinge (so more rigid). Binding of FGFR1 produced complexes that were less dissipative than the FGF-1 complexes. Intriguingly, deglycosylation of the FGFR1 increased the amount of binding to heparin tetrasaccharide, and this complex on a mass basis is more dissipative.
Conclusion   By using the QCM-D, we are able to gain insights into novel aspects of these protein-glycosaminoglycan complexes.     

The action of the NK1 tachykinin receptor antagonist, CP 99,994, in antagonizing the acute and delayed emesis induced by cisplatin in the ferret.

1. The anti-emetic effects of the NK1 tachykinin receptor antagonist, CP 99,994 (10 mg kg-1) were investigated in the ferret using a cisplatin-induced acute (day 1) and delayed (day 2 and 3) retching and vomiting model. 2. With a single cisplatin (10 mg kg-1) emetogenic challenge, the i.p. administration of CP 99,994 given as a single injection immediately following the first emetic episode, promptly abolished the retching and vomiting for a 4 h period. CP 99,994 was as efficacious as ondansetron (1.0 mg kg-1). The general toxicity of cisplatin 10 mg kg-1 precluded its use in studies of delayed emesis. 3. With a single cisplatin (5 mg kg-1) emetogenic challenge, the single administration of either CP 99,994 (10 mg kg-1) or ondansetron (1.0 mg kg-1) immediately following the first emetic episode markedly reduced or abolished the retching and vomiting for 4 h. Such single treatments failed to modify significantly the intensity of delayed emesis appearing on the second and third day. 4. With a cisplatin (5 mg kg-1) emetogenic challenge, administration of CP 99,994 (10 mg kg-1) at 8 hourly intervals, the first injection being administered 30 s post cisplatin, was associated with 4 or more abolitions of emesis during both the acute and delayed phase. A 4 hourly administration of CP 99,994 for 20 h during delayed emesis completely abolished the retching and vomiting. 5. It is concluded that cisplatin 5 mg kg-1 provides an emetogenic challenge causing an acute and delayed phase of retching and vomiting and that CP 99,994 can abolish both phases. The results may be relevant to the understanding and treatment of chemotherapy-induced emesis in man.     

HLA-C variants associated with amino acid substitutions in the peptide binding groove influence susceptibility to Kawasaki disease

Kawasaki disease (KD) is a pediatric vasculitis caused by an unknown trigger in genetically susceptible children. The incidence varies widely across genetically diverse populations. Several associations with HLA Class I alleles have been reported in single cohort studies. Using a genetic approach, from the nine single nucleotide variants (SNVs) associated with KD susceptibility in children of European descent, we identified SNVs near the HLA-C (rs6906846) and HLA-B genes (rs2254556) whose association was replicated in a Japanese descent cohort (rs6906846 p = 0.01, rs2254556 p = 0.005). The risk allele (A at rs6906846) was also associated with HLA-C*07:02 and HLA-C*04:01 in both US multi-ethnic and Japanese cohorts and HLA-C*12:02 only in the Japanese cohort. The risk A-allele was associated with eight non-conservative amino acid substitutions (amino acid positions); Asp or Ser (9), Arg (14), Ala (49), Ala (73), Ala (90), Arg (97), Phe or Ser (99), and Phe or Ser (116) in the HLA-C peptide binding groove that binds peptides for presentation to cytotoxic T cells (CTL). This raises the possibility of increased affinity to a “KD peptide” that contributes to the vasculitis of KD in genetically susceptible children.     

Refining the Primrose syndrome phenotype: A study of five patients with ZBTB20 de novo variants and a review of the literature

Primrose syndrome is a rare autosomal dominant condition caused by heterozygous missense variants within ZBTB20. Through an exome sequencing approach (as part of the Deciphering Developmental Disorders [DDD] study) we have identified five unrelated individuals with previously unreported, de novo ZBTB20 pathogenic missense variants. All five missense variants targeted the C2H2 zinc finger domains. This genotype‐up approach has allowed further refinement of the Primrose syndrome phenotype. Major characteristics (>90% individuals) include an intellectual disability (most frequently in the moderate range), a recognizable facial appearance and brain MRI abnormalities, particularly abnormalities of the corpus callosum. Other frequent clinical associations (in 50–90% individuals) include sensorineural hearing loss (83%), hypotonia (78%), cryptorchidism in males (75%), macrocephaly (72%), behavioral issues (56%), and dysplastic/hypoplastic nails (57%). Based upon these clinical data we discuss our current management of patients with Primrose syndrome.     

Deep phenotyping of 14 new patients with IQSEC2 variants,including monozygotic twins of discordant phenotype

Whole-exome sequencing has established IQSEC2 as a neurodevelopmental disability gene. The IQSEC2 variant phenotype includes developmental delay, intellectual disability, epilepsy, hypotonia, autism, developmental regression, microcephaly and stereotypies but is yet to be fully described. Presented here are 14 new patients with IQSEC2 variants. In addition to the established features, we observed: gait ataxia in 7 of 9 (77.8%), drooling in 9 of 14 (64.2%), early feeding difficulties in 7 of 14 (50%), structural brain abnormalities in 6 of 13 (46.2%), brachycephaly in 5 of 14 (35.7%), and scoliosis and paroxysms of laughter each in 4 of 14 (28.6%). We suggest that these are features of the IQSEC2-related disorder. Gastrostomy requirement, plagiocephaly, strabismus and cortical blindness, each seen in 2 of 14 (14.3%), may also be associated. Shared facial features were noted in 8 of 14 patients, and shared hair patterning was identified in 5 of 14 patients. This study further delineates the IQSEC2 phenotypic spectrum and supports the notion of an emerging IQSEC2 syndrome. We draw parallels between the IQSEC2-related disorder and the Angelman-/Rett-/Pitt-Hopkins syndrome group of conditions and recommend the addition of IQSEC2 to epilepsy and developmental delay gene panels. We observed discordant phenotypes in monozygotic twins and apparent gonadal mosaicism, which has implications for recurrence risk counselling in the IQSEC2-related disorder.