An IGF-I promoter polymorphism modifies the relationships between birth weight and risk factors for cardiovascular disease and diabetes at age 36

BACKGROUND: Biochemical testing for pheochromocytoma by measurement of fractionated plasma metanephrines is limited by false positive rates of up to 18% in people without known genetic predisposition to the disease. The plasma normetanephrine fraction is responsible for most false positives and plasma normetanephrine increases with age. The objective of this study was to determine if we could improve the specificity of fractionated plasma measurements, by statistically adjusting for age. METHODS: An age-adjusted metanephrine score was derived using logistic regression from 343 subjects (including 33 people with pheochromocytoma) who underwent fractionated plasma metanephrine measurements as part of investigations for suspected pheochromocytoma at Mayo Clinic Rochester (derivation set). The performance of the age-adjusted score was validated in a dataset of 158 subjects (including patients 23 with pheochromocytoma) that underwent measurements of fractionated plasma metanephrines at Mayo Clinic the following year (validation dataset). None of the participants in the validation dataset had known genetic predisposition to pheochromocytoma. RESULTS: The sensitivity of the age-adjusted metanephrine score was the same as that of traditional interpretation of fractionated plasma metanephrine measurements, yielding a sensitivity of 100% (23/23, 95% confidence interval [CI] 85.7%, 100%). However, the false positive rate with traditional interpretation of fractionated plasma metanephrine measurements was 16.3% (22/135, 95% CI, 11.0%, 23.4%) and that of the age-adjusted score was significantly lower at 3.0% (4/135, 95% CI, 1.2%, 7.4%) (p < 0.001 using McNemar's test). CONCLUSION: An adjustment for age in the interpretation of results of fractionated plasma metanephrines may significantly decrease false positives when using this test to exclude sporadic pheochromocytoma. Such improvements in false positive rate may result in savings of expenditures related to confirmatory imaging.     

The zebrafish mutants dre, uki, and lep encode negative regulators of the hedgehog signaling pathway

Proliferation is one of the basic processes that control embryogenesis. To identify factors involved in the regulation of proliferation, we performed a zebrafish genetic screen in which we used proliferating cell nuclear antigen (PCNA) expression as a readout. Two mutants, hu418B and hu540A, show increased PCNA expression. Morphologically both mutants resembled the dre (dreumes), uki (ukkie), and lep (leprechaun) mutant class and both are shown to be additional uki alleles. Surprisingly, although an increased size is detected of multiple structures in these mutant embryos, adults become dwarfs. We show that these mutations disrupt repressors of the Hedgehog (Hh) signaling pathway. The dre, uki, and lep loci encode Su(fu) (suppressor of fused), Hip (Hedgehog interacting protein), and Ptc2 (Patched2) proteins, respectively. This class of mutants is therefore unique compared to previously described Hh mutants from zebrafish genetic screens, which mainly show loss of Hh signaling. Furthermore, su(fu) and ptc2 mutants have not been described in vertebrate model systems before. Inhibiting Hh activity by cyclopamine rescues uki and lep mutants and confirms the overactivation of the Hh signaling pathway in these mutants. Triple uki/dre/lep mutants show neither an additive increase in PCNA expression nor enhanced embryonic phenotypes, suggesting that other negative regulators, possibly Ptc1, prevent further activation of the Hh signaling pathway. The effects of increased Hh signaling resulting from the genetic alterations in the uki, dre, and lep mutants differ from phenotypes described as a result of Hh overexpression and therefore provide additional insight into the role of Hh signaling during vertebrate development.     

Identification and characterization of a novel outer membrane protein (OMP J) of Moraxella catarrhalis that exists in two major forms

Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fisher's exact test; P < 0.01). Experiments using two DeltaompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic DeltaompJ2 mutant (unpaired Student's t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.     

Nidovirus sialate-O-acetylesterases: evolution and substrate specificity of coronaviral and toroviral receptor-destroying enzymes

Many viruses achieve reversible attachment to sialic acid (Sia) by encoding envelope glycoproteins with receptor-binding and receptor-destroying activities. Toroviruses and group 2 coronaviruses bind to O-acetylated Sias, presumably via their spike proteins (S), whereas other glycoproteins, the hemagglutinin-esterases (HE), destroy Sia receptors by de-O-acetylation. Here, we present a comprehensive study of these enzymes. Sialate-9-O-acetylesterases specific for 5-N-acetyl-9-O-acetylneuraminic acid, described for bovine and human coronaviruses, also occur in equine coronaviruses and in porcine toroviruses. Bovine toroviruses, however, express novel sialate-9-O-acetylesterases, which prefer the di-O-acetylated substrate 5-N-acetyl-7(8),9-di-O-acetylneuraminic acid. Whereas most rodent coronaviruses express sialate-4-O-acetylesterases, the HE of murine coronavirus DVIM cleaves 9-O-acetylated Sias. Under the premise that HE specificity reflects receptor usage, we propose that two types of Sias serve as initial attachment factors for coronaviruses in mice. There are striking parallels between orthomyxo- and nidovirus biology. Reminiscent of antigenic shifts in orthomyxoviruses, rodent coronaviruses exchanged S and HE sequences through recombination to extents not appreciated before. As for orthomyxovirus reassortants, the fitness of nidovirus recombinant offspring probably depends both on antigenic properties and on compatibility of receptor-binding and receptor-destroying activities.     

Aberrant expression of caspase-14 in epithelial tumors

Cysteine-dependent aspartate-specific proteases (caspases) are the cellular executors of apoptosis. Caspase-14 is the most divergent member of the family of mammalian caspases and displays a variety of unique characteristics. It is expressed in a limited number of tissues and has the shortest amino acid sequence within the caspase protein family. During induction of apoptosis, it is not processed, whereas terminal differentiation in skin leads to cleavage of caspase-14. Here we show that 40% of lung squamous cell carcinomas, 22% of breast cancers, and about 80% of cervical carcinomas express caspase-14. Immunohistochemistry reveals that caspase-14 is localized in areas of ongoing differentiation close to necrotic sites but is not strictly associated with the differentiation markers keratin-1/-10. Caspase-14 is neither mutated nor alternatively spliced in the tumors analyzed. Furthermore, caspase-14 is not processed into a small and large subunit, a process critical for the proteolytic activation of known effector caspases. We conclude that conditions exist in tumors leading to re-expression of this normally silent gene.     

An inhibitor binding pocket distinct from the catalytic active site on human beta-APP cleaving enzyme

Beta-APP cleaving enzyme (BACE) is responsible for the first of two proteolytic cleavages of the APP protein that together lead to the generation of the Alzheimer's disease-associated Abeta peptide. It is widely believed that halting the production of Abeta peptide, by inhibition of BACE, is an attractive therapeutic modality for the treatment of Alzheimer's disease. BACE is an aspartyl protease, and there is significant effort in the pharmaceutical community to apply traditional design methods to the development of active site-directed inhibitors of this enzyme. We report here the discovery of a ligand binding pocket within the catalytic domain of BACE that is distinct from the enzymatic active site (i.e., an exosite). Peptides, initially identified from combinatorial phage peptide libraries, contain the sequence YPYF(I/L)P(L/I) and bind specifically to this exosite, even in the presence of saturating concentrations of active site-directed inhibitors. Binding of peptides to the BACE exosite leads to a concentration-dependent inhibition of proteolysis for APP-related, protein-based substrates of BACE. The discovery of this exosite opens new opportunities for the identification and development of novel and potentially selective small molecule inhibitors of BACE that act through exosite, rather than active site, binding interactions.     

Adenovirus-mediated hepatic overexpression of scavenger receptor class B type I accelerates chylomicron metabolism in C57BL/6J mice

The function of scavenger receptor class B type I (SR-BI) in mediating the selective uptake of HDL cholesteryl esters is well established. In SR-BI-deficient mice, we recently observed a delayed postprandial triglyceride (TG) response, suggesting an additional role for SR-BI in facilitating chylomicron (CM) metabolism. Here, we assessed the effect of adenovirus-mediated hepatic overexpression of SR-BI (Ad.SR-BI) in C57BL/6J mice on serum lipids and CM metabolism. Infection of 5 x 10(8) plaque-forming units per mouse of Ad.SR-BI significantly decreases serum cholesterol (>90%), phospholipids (>90%), and TG levels (50%), accompanied by a 41.4% reduction (P < 0.01) in apolipoprotein B-100 levels. The postprandial TG response is 2-fold lower in mice treated with Ad.SR-BI compared with control mice (area under the curve = 31.4 +/- 2.4 versus 17.7 +/- 3.2; P < 0.05). Hepatic mRNA expression levels of genes known to be involved in serum cholesterol and TG clearance are unchanged and thus could not account for the decreased plasma TG levels and the change in postprandial response. We conclude that overexpression of SR-BI accelerates CM metabolism, possibly by mediating the initial capture of CM remnants by the liver, whereby the subsequent internalization can be exerted by additional receptor systems such as the LDL receptor (LDLr) and LDLr-related protein 1.     

The fate of sheep-dispersed seeds: Plant species emergence and spatial patterns

Sheep epizoochory has often been proposed as an important vector which can help to overcome the dispersal limitation of plants in fragmented landscapes. In order to evaluate the contribution of herbivores to recruitment especially of target species, the dispersal and post-dispersal fate of such seeds must be known. In a field experiment sheep with seeds of mainly target species (experimentally attached to their coats) were present at three sand plots for 24 h. Natural epizoochorous dispersal was already shown for most of the species in our study area. Seed detachment, trampling intensity and seed shadow were measured; seedling emergence and survival were recorded over an 8-month period. In addition, the effect of sheep trampling on seedling emergence and survival of two threatened species, Jurinea cyanoides and Koeleria glauca, were studied.     

Identification of novel allosteric nonpeptidergic inhibitors of the human cytomegalovirus-encoded chemokine receptor US28

Human cytomegalovirus (HCMV) is a widespread human pathogen, possessing onco-modulatory properties. Constitutive signaling of the HCMV-encoded chemokine receptor US28 and its ability to bind a broad spectrum of chemokines might facilitate HCMV-associated tumor progression. Novel nonpeptidergic chemotypes were identified as neutral antagonists or inverse agonists on US28, that allosterically inhibit chemokine binding to US28.