Studies on sphingomyelinase and beta-glucosidase activities in Niemann-Pick disease variants. Phosphodiesterase activities measured with natural and artificial substrates

Cultured fibroblasts were studied from 12 cases of Niemann-Pick disease group C. In 11, sphingomyelinase and glucocerebrosidase (and beta-glucosidase) activities were reduced to around 50% of those of controls. On isoelectric focusing, all 12 strains lacked sphingomyelinase activity in the major cathodic region (pI 8.0). The defect was also demonstrated with the artificial phosphodiester substrates bis(4-methylumbelliferyl) phosphate and 4-methylumbelliferyl pyrophosphate diester. In control fibroblasts and those heterozygous for types A or B or group C Niemann-Pick disease, the major sphingomyelinase peak electrofocused at pI 8.0. No direct interaction could be demonstrated by mixing experiments between group C Niemann-Pick extracts and those of type A disease or Gaucher disease. Profiles for beta-glucosidase activity appeared normal in Niemann-Pick group C fibroblasts. No reduction of sphingomyelinase or glucocerebrosidase activities was found in Niemann-Pick group C liver, nor any attenuation of cathodic sphingomyelinase activity in the affected tissue. Results suggest that sphingomyelinase expression differs in fibroblasts and liver. Enzyme defects associated with Niemann-Pick disease group C were only observed in cultured cells.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

A role for the phosphorylation of hRad9 in checkpoint signaling

The integrity of the human genome is preserved by signal transduction pathways called checkpoints, which delay progression through the cell cycle when DNA damage is present. Three checkpoint proteins, hRad9, hRad1, and hHus1, form a proliferating cell nuclear antigen-like, heterotrimeric complex that has been proposed to function in the initial detection of DNA structural abnormalities. hRad9 is highly modified by phosphorylation, in a constitutive manner and in response to both DNA damage and cell cycle position. Here we present evidence that Thr292 of hRad9 is subject to Cdc2-dependent phosphorylation in mitosis. Furthermore, our data are also consistent with four other hRad9 phosphorylation sites (Ser277, Ser328, Ser336, and Thr355) being regulated in part by Cdc2. We also identify Ser387 as a novel site of hRad9 constitutive phosphorylation and show that phosphorylation at Ser387 is a prerequisite for one form of DNA damage-induced hyperphosphorylation of hRad9. Characterization of nonphosphorylatable mutants has revealed that hRad9 phosphorylation plays a critical role in checkpoint signaling. Overexpression of these mutants blocks the interaction between hRad9 and the DNA damage-responsive protein TopBP1 and impairs the cellular response to DNA damage during S phase.     

Utility of the white gene in estimating phylogenetic relationships among mosquitoes (Diptera: Culicidae)

The utility of a nuclear protein-coding gene for reconstructing phylogenetic relationships within the family Culicidae was explored. Relationships among 13 species representing three subfamilies and nine genera of Culicidae were analyzed using a 762-bp fragment of coding sequence from the eye color gene, white. Outgroups for the study were two species from the sister group Chaoboridae. Sequences were determined from clone PCR products amplified from genomic DNA, and aligned following conceptual intron splicing and amino acid translation. Third codon positions were characterized by high levels of divergence and biased nucleotide composition, the intensity and direction of which varied among taxa. Equal weighting of all characters resulted in parsimony and neighboring-joining trees at odds with the generally accepted phylogenetic hypothesis based on morphology and rDNA sequences. The application of differential weighting schemes recovered the traditional hypothesis, in which the subfamily Anophelinae formed the basal clade. The subfamily Toxorhynchitinae occupied an intermediate position, and was a sister group to the subfamily Culicinae. Within Culicinae, the genera Sabethes and Tripteroides formed an ancestral clade, while the Culex-Deinocerites and Aedes- Haemagogus clades occupied increasingly derived positions in the molecular phylogeny. An intron present in the Culicinae- Toxorhynchitinae lineage and one outgroup taxon was absent in the basal Anophelinae lineage and the second outgroup taxon, suggesting that intron insertions or deletions may not always be reliable systematic characters.      

Post-translational fate of variant MOPC 315 lambda chains in Xenopus oocytes and mouse myeloma cells

The post-translational fates of three immunoglobulin lambda chain variants of MOPC 315 were investigated in mouse plasmacytoma cell lines and in mRNA-microinjected Xenopus oocytes. Quite unexpectedly we found that one non-secretory variant chain (lambda-43) underwent extensive post-translational N-glycosylation: however the presence of the oligosaccharide moiety did not account for the nonsecretory phenotype nor did it affect the rate of degradation of this lambda chain. Another variant chain (lambda-47) at first believed to be non-secretory, was found to be secreted from oocytes at a very low level, but mostly as a lambda-lambda dimer. In myeloma cells a low level of lambda-47 chain was secreted and again lambda-lambda dimers were the favoured secretory form. The secretory lambda-48 chain also formed lambda-lambda dimers, whereas lambda-43, which was never secreted, was only found as a monomeric lambda chain in both oocytes and myeloma cells. A similar relationship between assembly and secretion was found when oocytes were coinjected with MOPC 21 heavy (gamma 1) chain mRNA and MOPC 315 lambda chain mRNAs. The wild type lambda chain (lambda-48) was able to assemble with the gamma chain in a covalently bound tetramer (gamma gamma lambda lambda). The variant lambda-47 chain was also able to form gamma gamma lambda lambda tetramers, whereas the lambda-43 was not, even when glycosylation was prevented by tunicamycin. Both types of tetramer were secreted. These data reinforce the idea that conformational changes play a major role in the routing of secretory proteins and that the cellular mechanisms by which these changes are recognized are not cell-type specific.     

Somatic cell hybridisation studies showing different gene mutations in Niemann-Pick variants

Summary Cultured skin fibroblasts from patients with different clinical types of Niemann-Pick disease were hybridized and sphingomyelinase activities were measured in the heterokaryon cell population. Both the natural substrate (³H-choline) sphingomyelin and the chromogenic analogue hexadecanoylamino-4-nitrophenylphosphorylcholine were used in the complementation analysis. In fusions between cells from type C Niemann-Pick disease with those from type A or B a clear restoration of sphingomyelinase activity occurred, whereas no complementation was found in other fusion combinations. The results indicate that at least two different genes are involved in the mutations leading to the different Niemann-Pick variants.     

Compound heterozygosity for a Wolman mutation is frequent among patients with cholesteryl ester storage disease

Cholesteryl ester storage disease and Wolman disease are rare autosomal recessive lipoprotein-processing disorders caused by mutations in the gene encoding human lysosomal acid lipase. Thus far we have elucidated the genetic defects in 15 unrelated CESD patients. Seven were homozygotes for the prevalent hLAL exon 8 splice junction mutation which results in incomplete exon skipping, while eight probands were compound heterozygotes for E8SJM and a rare mutation on the second chromosome. In this report, we describe the molecular basis of CESD in three compound heterozygous subjects of Czech and Irish origin. RFLP and DNA sequence analysis revealed that they were heteroallelic for the common G(934)-->A substitution in exon 8 of the hLAL gene and a mutation which, if inherited on both alleles, would be expected to result in complete loss of enzyme activity and to cause Wolman disease. In patients A. M. and J. J., two nucleotide deletions in exons 7 and 10 were detected, involving a T at position 722, 723, or 724 and a G in a stretch of five guanosines at positions 1064;-1068 of the hLAL cDNA. Both mutations result in premature termination of protein translation at residues 219 and 336, respectively, and in the production of truncated, inactive enzymes. Subject D. H., in contrast, is a compound heterozygote for the Arg(44)-->Stop mutation previously described in a French CESD proband. Combined with data in the literature, our results demonstrate that compound heterozygosity for a mutation causing Wolman disease is common among cholesteryl ester storage disease patients.     

ImmunoPET and biodistribution with human epidermal growth factor receptor 3 targeting antibody 89Zr-RG7116

The humanized monoclonal antibody with high affinity for the human epidermal growth factor receptor (HER) 3, RG7116, is a glycoengineered, IgG1 class antibody. By labeling RG7116 with zirconium-89 (⁸⁹Zr) we aimed to visualize in vivo HER3 expression and study the biodistribution of this antibody in human tumor-bearing mice. Biodistribution of ⁸⁹Zr-RG7116 was studied in subcutaneously xenografted FaDu tumor cells (HER3-positive). Dose-dependency of ⁸⁹Zr-RG7116 organ distribution and specific tumor uptake was assessed by administering doses ranging from 0.05 to 10 mg/kg RG7116 to SCID/Beige mice. Biodistribution was analyzed at 24 and 144 h after injection. MicroPET imaging was performed at 1, 3, and 6 days after injection of 1.0 mg/kg ⁸⁹Zr-RG7116 in the FaDu, H441, QG-56 and Calu-1 xenografts with varying HER3 expression. The excised tumors were analyzed for HER3 expression. Biodistribution analyses showed a dose- and time-dependent ⁸⁹Zr-RG7116 tumor uptake in FaDu tumors. The highest tumor uptake of ⁸⁹Zr-RG7116 was observed in the 0.05 mg/kg dose group with 27.5%ID/g at 144 h after tracer injection. MicroPET imaging revealed specific tumor uptake of ⁸⁹Zr-RG7116 in FaDu and H441 models with an increase in tumor uptake over time. Biodistribution data was consistent with the microPET findings in FaDu, H441, QG56 and Calu-1 xenografts, which correlated with HER3 expression levels. In conclusion, ⁸⁹Zr-RG7116 specifically accumulates in HER3 expressing tumors. PET imaging with this tracer provides real-time non-invasive information about RG7116 distribution, tumor targeting and tumor HER3 expression levels.