Selective incorporation of polyanionic molecules into hamster prions

The central pathogenic event of prion disease is the conformational conversion of a host protein, PrPC, into a pathogenic isoform, PrPSc. We previously showed that the protein misfolding cyclic amplification (PMCA) technique can be used to form infectious prion molecules de novo from purified native PrPC molecules in an autocatalytic process requiring accessory polyanions (Deleault, N. R., Harris, B. T., Rees, J. R., and Supattapone, S. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 9741-9746). Here we investigated the molecular mechanism by which polyanionic molecules facilitate infectious prion formation in vitro.Ina PMCA reaction lacking PrPSc template seed, synthetic polyA RNA molecules induce hamster HaPrPC to adopt a protease-sensitive, detergent-insoluble conformation reactive against antibodies specific for PrPSc. During PMCA, labeled nucleic acids form nuclease-resistant complexes with HaPrP molecules. Strikingly, purified HaPrPC molecules subjected to PMCA selectively incorporate an approximately 1-2.5-kb subset of [32P]polyA RNA molecules from a heterogeneous mixture ranging in size from approximately 0.1 to >6 kb. Neuropathological analysis of scrapie-infected hamsters using the fluorescent dye acridine orange revealed that RNA molecules co-localize with large extracellular HaPrP aggregates. These findings suggest that polyanionic molecules such as RNA may become selectively incorporated into stable complexes with PrP molecules during the formation of native hamster prions.     

Metabolic engineering of the baculovirus-expression system via inverse "shotgun" genomic analysis and RNA interference (dsRNA) increases product yield and cell longevity

RNA interference (RNAi) is as powerful tool for characterizing gene function in eukaryotic organisms and cultured cell lines. Its use in metabolic engineering has been limited and few reports have targeted protein expression systems to increase yield. In this work, we examine the use of in vitro synthesized double stranded RNA (dsRNA) in the baculovirus expression vector system (BEVS), using commercially relevant cultured cells (Spodoptera frugiperda, Sf-9) and larvae (Trichoplusia ni) as hosts. First, we employed an inverse "shotgun" genomic analysis to "find" an array of 16 putative insect gene targets. We then synthesized dsRNA in vitro targeting these genes and investigated the effects of injected dsRNA on larval growth, development, and product yield. Growth and development was at times stunted and in several cases, the effects were lethal. However, dsRNA targeting an acidic juvenile hormone-suppressible protein (AJHSP1), and translational elongation factor 2 (Ef-2) resulted in significantly increased yield of model product, GFP. Next, we targeted known genes, v-cath and apoptosis inducer, sf-caspase 1, in cultured Sf-9 cells. We confirm RNAi-mediated sf-caspase 1 suppression in Sf-9 cells, but not in baculovirus-infected cells, likely due to the overriding effects of inhibitor of apoptosis protein, p35. We also demonstrate suppression of v-cath in infected cells, which leads to a approximately 3-fold increase in product yield. Overall, our results support the application of RNAi in metabolic engineering, specifically for enhancing protein productivity in the baculovirus expression vector system.     

Association between ultrasound-detected synovitis and knee pain: a population-based case–control study with both cross-sectional and follow-up data

Background

An important role for synovial pathology in the initiation and progression of knee osteoarthritis has been emphasised recently. This study aimed to examine whether ultrasonography-detected synovial changes associate with knee pain (KP) in a community population.

Methods

A case–control study was conducted to compare people with early KP (n?=?298), established KP (n?=?100) or no KP (n?=?94) at baseline. Multinomial logistic regression was used to estimate the odds ratio (OR) and 95% confidence interval (CI) between groups adjusted for radiographic osteoarthritis (ROA) severity and other confounding factors. After 1 year, 255 participants with early and established KP completed the follow-up questionnaire for changes in KP. Logistic regression with adjustment was used to determine predictors of KP worsening.

Results

At baseline, effusion was associated with early KP (OR 2.64, 95% CI 1.57–4.45) and established KP (OR 5.07, 95% CI 2.74–9.38). Synovial hypertrophy was also associated with early KP (OR 5.43, 95% CI 2.12–13.92) and established KP (OR 13.27, 95% CI 4.97–35.43). The association with effusion diminished when adjusted for ROA. Power Doppler signal was uncommon (early KP 3%, established KP 2%, controls 0%). Baseline effusion predicted worsening of KP at 1 year (OR 1.95, 95% CI 1.05–3.64). However, after adjusting for ROA, the prediction was insignificant (adjusted OR 0.95, 95% CI 0.44–2.02).

Conclusions

Ultrasound effusion and synovial hypertrophy are associated with KP, but only effusion predicts KP worsening. However, the association/prediction is not independent from ROA. Power Doppler signal is uncommon in people with KP. Further study is needed to understand whether synovitis is directly involved in different types of KP.

     

Vitamin D substrate–product relationship in idiopathic hypercalciuria

Absorptive hypercalciuria (AH) is associated with elevated levels of 1,25-dihydroxyvitamin D (1,25(OH)₂D). While no increase of 1,25(OH)₂D after oral administration of 25-hydroxyvitamin D (25OHD) at high doses has been claimed in normal subjects, a substrate–product relationship has been reported in normal children, young people after UV irradiation, older persons, postmenopausal women, primary hyperparathyroidism, renal failure, osteomalacia, and sarcoidosis. No data of this relationship in AH is available. To investigate 25OHD-1,25(OH)₂D substrate–product relationship in AH, 161 AH patients (mean age 60.9 ± 11.7 years) and 110 age- and sex-matched controls (mean age 61.5 ± 12.4 years) were studied. In 57 controls and 52 AH subjects 25OHD-1,25(OH)₂D relationship in basal conditions and after 2-week oral 25OHD (25 μg/day) administration were evaluated. In basal conditions 25OHD and 1,25(OH)₂D were correlated in both, controls and AH; 25OHD treatment was followed by an increase in serum 25OHD and 1,25(OH)₂D in both groups. However, delta responses of 25OHD and 1,25(OH)₂D to 25OHD were higher in AH suggesting an enhanced activity of 1α-hydroxylase. In conclusion, the higher response of 1,25(OH)₂D after oral 25OHD in AH patients suggests a differential capacity between both groups in handling the increases in 1,25(OH)₂D.     

The Pharmacogenetic Footprint of ACE Inhibition: A Population-Based Metabolomics Study

Angiotensin-I-converting enzyme (ACE) inhibitors are an important class of antihypertensives whose action on the human organism is still not fully understood. Although it is known that ACE especially cleaves COOH-terminal dipeptides from active polypeptides, the whole range of substrates and products is still unknown. When analyzing the action of ACE inhibitors, effects of genetic variation on metabolism need to be considered since genetic variance in the ACE gene locus was found to be associated with ACE-concentration in blood as well as with changes in the metabolic profiles of a general population. To investigate the interactions between genetic variance at the ACE-locus and the influence of ACE-therapy on the metabolic status we analyzed 517 metabolites in 1,361 participants from the KORA F4 study. We replicated our results in 1,964 individuals from TwinsUK. We observed differences in the concentration of five dipeptides and three ratios of di- and oligopeptides between ACE inhibitor users and non-users that were genotype dependent. Such changes in the concentration affected major homozygotes, and to a lesser extent heterozygotes, while minor homozygotes showed no or only small changes in the metabolite status. Two of these resulting dipeptides, namely aspartylphenylalanine and phenylalanylserine, showed significant associations with blood pressure which qualifies them—and perhaps also the other dipeptides—as readouts of ACE-activity. Since so far ACE activity measurement is substrate specific due to the usage of only one oligopeptide, taking several dipeptides as potential products of ACE into account may provide a broader picture of the ACE activity.     

Genetic influences on female infidelity and number of sexual partners in humans: a linkage and association study of the role of the vasopressin receptor gene (AVPR1A).

In humans, in contrast to animals, the genetic influences on infidelity are unclear. We report here a large study of over 1600 unselected United Kingdom female twin pairs who confidentially reported previous episodes of infidelity and total lifetime number of sexual partners, as well as attitudes towards infidelity. Our findings demonstrate that infidelity and number of sexual partners are both under moderate genetic influence (41% and 38% heritable, respectively) and the genetic correlation between these two traits is strong (47%). Conversely, attitudes towards infidelity are driven by shared and unique environmental, but not genetic, influences. A genome-wide linkage scan identified three suggestive but nonsignificant linkage areas associated with infidelity and number of sexual partners on chromosomes 3, 7 and 20 with a maximum LOD score of 2.46. We were unsuccessful in associating infidelity or number of sexual partners with a locus implicated in other mammals' sexual behavior, the vasopressin receptor gene. Nonetheless, our findings on the heritability of sexual infidelity and number of sexual partners provide support for certain evolutionary theories of human sexual behavior, as well as justifying further genetic and molecular research in this domain.     

Effects of receptor concentration, media pH and storage on nicotinic receptor-transmitted signal in a fiber-optic biosensor

The optical signal generated by a nicotinic acetylcholine receptor (nAChR)-based optical biosensor was dependent on the density of toxin binding sites (i.e. receptors) immobilized on the surface of the fiber. The maximum density of nAChR receptors absorbed on the optic fibers, measured using [125I]-alpha-bungarotoxin (the alpha-neurotoxin of the Bungarus snake venom) binding, was 6.2 pmol of receptor sites per fiber. Incubation time, that was required for maximal noncovalent immobilization of the receptor protein on the fiber, was less than 10 min. Immobilization of the nAChR protein on the quartz fiber was affected by pH of the medium, with pH 3.5 as the optimal. Stored in phosphate buffered saline (PBS) or in a dry nitrogen atmosphere, the nAChR optical sensor showed no loss of activity over the first 3 days, then showed a slow but gradual loss in activity (45% over the next 30 days).