QTL mapping for genetic determinants of lipoprotein cholesterol levels in combined crosses of inbred mouse strains

To identify additional loci that influence lipoprotein cholesterol levels, we performed quantitative trait locus (QTL) mapping in offspring of PERA/EiJxI/LnJ and PERA/EiJxDBA/2J intercrosses and in a combined data set from both crosses after 8 weeks of consumption of a high fat-diet. Most QTLs identified were concordant with homologous chromosomal regions that were associated with lipoprotein levels in human studies. We detected significant new loci for HDL cholesterol levels on chromosome (Chr) 5 (Hdlq34) and for non-HDL cholesterol levels on Chrs 15 (Nhdlq9) and 16 (Nhdlq10). In addition, the analysis of combined data sets identified a QTL for HDL cholesterol on Chr 17 that was shared between both crosses; lower HDL cholesterol levels were conferred by strain PERA. This QTL colocalized with a shared QTL for cholesterol gallstone formation detected in the same crosses. Haplotype analysis narrowed this QTL, and sequencing of the candidate genes Abcg5 and Abcg8 confirmed shared alleles in strains I/LnJ and DBA/2J that differed from the alleles in strain PERA/EiJ. In conclusion, our analysis furthers the knowledge of genetic determinants of lipoprotein cholesterol levels in inbred mice and substantiates the hypothesis that polymorphisms of Abcg5/Abcg8 contribute to individual variation in both plasma HDL cholesterol levels and susceptibility to cholesterol gallstone formation.     

Crystal structure of rubredoxin from Pyrococcus furiosus at 0.95 Å resolution, and the structures of N-terminal methionine and formylmethionine variants of Pf Rd. Contributions of N-terminal interactions to thermostability

The high-resolution crystal structure of the small iron-sulfur protein rubredoxin (Rd) from the hyperthermophilic archeon Pyrococcus furiosus (Pf) is reported in this paper, together with those of its methionine ([_0M]Pf Rd) and formylmethionine (f[_0M]Pf Rd) variants. These studies were conducted to assess the consequences of the presence or absence of a salt bridge between the amino terminal nitrogen of Ala1 and the side chain of Glu14 to the structure and stability of this rubredoxin. The structure of wild-type Pf Rd was solved to a resolution of 0.95?Å and refined by full-matrix least-squares techniques to a crystallographic agreement factor of 12.8% [F>2σ(F) data, 25?617 reflections], while those of the [_0M]Pf and f[_0M]Pf Rd variants were solved at slightly lower resolutions (1.1?Å, R=11.5%, 17?213 reflections; 1.2?Å, R=13.7%, 12?478 reflections, respectively). The quality of the data was such that about half of the hydrogen atoms of the protein were clearly visible. All three structures were ultimately refined using the program SHELXL-93 with anisotropic atomic displacement parameters for all non-hydrogen protein atoms, and calculated hydrogen positions included but not refined. In this paper we also report thermostability data for all three forms of Pf Rd, and show that they follow the sequence wild-type >[_0M]Pf>formyl[_0M]Pf. Comparison of the three Pf Rd structures in the N-terminal region show that the structures of wild-type Pf Rd and f[_0M]Pf are rather similar, while that of [_0M]Pf Rd shows a number of additional hydrogen bonds involving the extra methionine group. While the salt bridge between the Ala1 amino group and the Glu14 carboxylate is not the primary determinant of the thermostability of Pf Rd, alterations to the amino terminus do have a moderate influence on the thermostability of this protein.     

Developmental refinement of hair cell synapses tightens the coupling of Ca2+ influx to exocytosis

Cochlear inner hair cells (IHCs) develop from pre‐sensory pacemaker to sound transducer. Here, we report that this involves changes in structure and function of the ribbon synapses between IHCs and spiral ganglion neurons (SGNs) around hearing onset in mice. As synapses matured they changed from holding several small presynaptic active zones (AZs) and apposed postsynaptic densities (PSDs) to one large AZ/PSD complex per SGN bouton. After the onset of hearing (i) IHCs had fewer and larger ribbons; (ii) Ca_V1.3 channels formed stripe‐like clusters rather than the smaller and round clusters at immature AZs; (iii) extrasynaptic Ca_V1.3‐channels were selectively reduced, (iv) the intrinsic Ca²⁺ dependence of fast exocytosis probed by Ca²⁺ uncaging remained unchanged but (v) the apparent Ca²⁺ dependence of exocytosis linearized, when assessed by progressive dihydropyridine block of Ca²⁺ influx. Biophysical modeling of exocytosis at mature and immature AZ topographies suggests that Ca²⁺ influx through an individual channel dominates the [Ca²⁺] driving exocytosis at each mature release site. We conclude that IHC synapses undergo major developmental refinements, resulting in tighter spatial coupling between Ca²⁺ influx and exocytosis.     

IL-23 p19 Knockout Mice Exhibit Minimal Defects in Responses to Primary and Secondary Infection with Francisella tularensis LVS

Our laboratory’s investigations into mechanisms of protective immunity against Francisella tularensis Live Vaccine Strain (LVS) have uncovered mediators important in host defense against primary infection, as well as those correlated with successful vaccination. One such potential correlate was IL-12p40, a pleiotropic cytokine that promotes Th1 T cell function as part of IL-12p70. LVS-infected IL-12p40 deficient knockout (KO) mice maintain a chronic infection, but IL-12p35 KO mice clear LVS infection; thus the role that IL-12p40 plays in immunity to LVS is independent of the IL-12p70 heterodimer. IL-12p40 can also partner with IL-23p19 to create the heterodimeric cytokine IL-23. Here, we directly tested the role of IL-23 in LVS resistance, and found IL-23 to be largely dispensable for immunity to LVS following intradermal or intranasal infection. IL-23p19 KO splenocytes were fully competent in controlling intramacrophage LVS replication in an in vitro overlay assay. Further, antibody responses in IL-23p19 KO mice were similar to those of normal wild type mice after LVS infection. IL-23p19 KO mice or normal wild type mice that survived primary LVS infection survived maximal doses of LVS secondary challenge. Thus p40 has a novel role in clearance of LVS infection that is unrelated to either IL-12 or IL-23.     

Nilotinib Is More Potent than Imatinib for Treating Plexiform Neurofibroma In Vitro and In Vivo

Plexiform neurofibromas (PNFs) are benign nerve sheath tumors mostly associated with neurofibromatosis type 1. They often extend through multiple layers of tissue and therefore cannot be treated satisfactorily by surgery. Nilotinib is a tyrosine kinase inhibitor used to treat leukemia, with advantages over the prototype imatinib in terms of potency and selectivity towards BCR-ABL, and the DDR, PDGFR, and KIT receptor kinases. In this study, we compared efficacies of the two drugs on cultured cells of PNF in vitro and on xenografted tumor fragments on sciatic nerve of athymic nude mice. Xenografts were monitored weekly using a high resolution ultrasound measurement. Treatment with nilotinib at a daily dose of 100 mg/kg for four weeks led to a reduction of the graft sizes_std by 68_±7% in the 8 treated mice, significantly more than the 33_±8% reduction in the 8 untreated mice (P<0.05) and the 47_±15% in the 7 mice treated with imatinib (P<0.05). The peak plasma nilotinib concentration 6.6_±1.1 µM is within the pharmacological range of clinical application. Imatinib, but not nilotinib significantly hindered body weight increase of the mice and elevated cytotoxicity of mouse spleen cells (P<0.05). Our results suggest that nilotinib may be more potent than imatinib for treating PNFs and may also be better tolerated. Imatinib seems to have some off-target effect in elevating immunity.     

Isolation and Partial Characterization of a Staphylococcal Leukocyte Cytotaxin

A factor which attracts rabbit polymorphonuclear leukocytes both in vitro and in vivo was isolated from the culture filtrate of a strain of Staphylococcus aureus. The activity of the chemotactic factor was independent of fresh serum and it was nondialyzable. Incubation of the factor with heat-inactivated human serum markedly inhibited its chemotactic property. The factor was heat-labile (80 C, 10 min) in the crude culture filtrate but was heat-stable when partially purified.     

Protein phosphatase 2A (PP2A)-specific ubiquitin ligase MID1 is a sequence-dependent regulator of translation efficiency controlling 3-phosphoinositide-dependent protein kinase-1 (PDPK-1)

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells.     

Effects of genetic variants in ADCY5, GIPR, GCKR and VPS13C on early impairment of glucose and insulin metabolism in children

Objective

Recent genome-wide association studies identified novel candidate genes for fasting and 2 h blood glucose and insulin levels in adults. We investigated the role of four of these loci (ADCY5, GIPR, GCKR and VPS13C) in early impairment of glucose and insulin metabolism in children.

Research Design and Methods

We genotyped four variants (rs2877716; rs1260326; rs10423928; rs17271305) in 638 Caucasian children with detailed metabolic testing including an oGTT and assessed associations with measures of glucose and insulin metabolism (including fasting blood glucose, insulin levels and insulin sensitivity/secretion indices) by linear regression analyses adjusted for age, sex, BMI-SDS and pubertal stage.

Results

The major allele (C) of rs2877716 (ADCY5) was nominally associated with decreased fasting plasma insulin (P = 0.008), peak insulin (P = 0.009) and increased QUICKI (P = 0.016) and Matsuda insulin sensitivity index (P = 0.013). rs17271305 (VPS13C) was nominally associated with 2 h blood glucose (P = 0.009), but not with any of the insulin or insulin sensitivity parameters. We found no association of the GIPR and GCKR variants with parameters of glucose and insulin metabolism. None of the variants correlated with anthropometric traits such as height, WHR or BMI-SDS, which excluded potential underlying associations with obesity.

Conclusions

Our data on obese children indicate effects of genetic variation within ADCY5 in early impairment of insulin metabolism and VPS13C in early impairment of blood glucose homeostasis.