Effects of 5' regulatory-region polymorphisms on paraoxonase-gene (PON1) expression

Human HDL-associated paraoxonase (PON1) hydrolyzes a number of toxic organophosphorous compounds and reduces oxidation of LDLs and HDLs. These properties of PON1 account for its ability to protect against pesticide poisonings and atherosclerosis. PON1 also hydrolyzes a number of lactone and cyclic-carbonate drugs. Among individuals in a population, PON1 levels vary widely. We previously identified three polymorphisms in the PON1 regulatory region that affect expression levels in cultured human hepatocytes. In this study, we determined the genotypes of three regulatory-region polymorphisms for 376 white individuals and examined their effect on plasma-PON1 levels, determined by rates of phenylacetate hydrolysis. The -108 polymorphism had a significant effect on PON1-activity level, whereas the -162 polymorphism had a lesser effect. The -909 polymorphism, which is in linkage disequilibrium with the other sites, appears to have little or no independent effect on PON1-activity level in vivo. Other studies have found that the L55M polymorphism in the PON1-coding region is associated with differences in both PON1-mRNA and PON1-activity levels. The results presented here indicate that the L55M effect of lowered activity is not due to the amino acid change but is, rather, largely due to linkage disequilibrium with the -108 regulatory-region polymorphism. The codon 55 polymorphism marginally appeared to account for 15.3% of the variance in PON1 activity, but this dropped to 5% after adjustments for the effects of the -108 and Q192R polymorphisms were made. The -108C/T polymorphism accounted for 22.8% of the observed variability in PON1-expression levels, which was much greater than that attributable to the other PON1 polymorphisms. We also identified four sequence differences in the 3' UTR of the PON1 mRNA.     

Induction of CD1-restricted immune responses in guinea pigs by immunization with mycobacterial lipid antigens

Group 1 CD1 molecules have been shown to present lipid and glycolipid Ags of mycobacteria to human T cells. However, a suitable animal model for the investigation of this component of antimycobacterial immunity has not yet been established. Previously, we found that guinea pigs express multiple isoforms of group 1 CD1 proteins that are homologous to human CD1b and CD1c. In this study, we show that CD1-restricted T cell responses can be generated in guinea pigs following immunization with lipid Ags from Mycobacterium tuberculosis. Splenic T cells from lipid Ag-immunized guinea pigs showed strong proliferative responses to total lipid Ags and partially purified glycolipid fractions from M. tuberculosis. These lipid Ag-reactive T cells were enriched in CD4-negative T cell fractions and showed cytotoxic activity against CD1-expressing guinea pig bone marrow-derived dendritic cells pulsed with M. tuberculosis lipid Ags. Using guinea pig cell lines transfected with individual CD1 isoforms as target cells in cytotoxic T cell assays, we found that guinea pig CD1b and CD1c molecules presented M. tuberculosis glycolipid Ags to T cells raised by mycobacterial lipid immunization. These results were confirmed using a T cell line derived from M. tuberculosis lipid Ag-immunized guinea pigs, which also showed CD1-restricted responses and cytolytic activity. Our results demonstrate that CD1-restricted responses against microbial glycolipid Ags can be generated in vivo by specific immunization and provide support for the use of the guinea pig as a relevant small animal model for the study of CD1-restricted immune responses to mycobacterial pathogens.     

The size of lipid rafts: an atomic force microscopy study of ganglioside GM1 domains in sphingomyelin/DOPC/cholesterol membranes

Atomic force microscopy has been used to study the distribution of ganglioside GM1 in model membranes composed of ternary lipid mixtures that mimic the composition of lipid rafts. The results demonstrate that addition of 1% GM1 to 1:1:1 sphingomyelin/dioleoylphosphatidylcholine/cholesterol monolayers leads to the formation of small ganglioside-rich microdomains (40-100 nm in size) that are localized preferentially in the more ordered sphingomyelin/cholesterol-rich phase. With 5% GM1 some GM1 microdomains are also detected in the dioleoylphosphatidylcholine-rich phase. A similar preferential localization of GM1 in the ordered phase is observed for bilayers with the same ternary lipid mixture in the upper leaflet. The small GM1-rich domains observed in these experiments are similar to the sizes for lipid rafts in natural membranes but considerably smaller than the ordered bilayer domains that have been shown to be enriched in GM1 in recent fluorescence microscopy studies of lipid bilayers. The combined data from a number of studies of model membranes indicate that lateral organization occurs on a variety of length scales and mimics many of the properties of natural membranes.     

Central neural mechanisms mediating human visceral hypersensitivity

Although visceral hypersensitivity is thought to be important in generating symptoms in functional gastrointestinal disorders, the neural mechanisms involved are poorly understood. We recently showed that central sensitization (hyperexcitability of spinal cord sensory neurones) may play an important role. In this study, we demonstrate that after a 30-min infusion of 0.15 M HCl acid into the healthy human distal esophagus, we see a reduction in the pain threshold to electrical stimulation of the non-acid-exposed proximal esophagus (9.6 +/- 2.4 mA) and a concurrent reduction in the latency of the N1 and P2 components of the esophageal evoked potentials (EEP) from this region (10.4 +/- 2.3 and 15.8 +/- 5.3 ms, respectively). This reduced EEP latency indicates a central increase in afferent pathway velocity and therefore suggests that hyperexcitability within the central visceral pain pathway contributes to the hypersensitivity within the proximal, non-acid-exposed esophagus (secondary hyperalgesia/allodynia). These findings provide the first electrophysiological evidence that central sensitization contributes to human visceral hypersensitivity.     

A cortical evoked potential study of afferents mediating human esophageal sensation

The aim of this study was to compare the characteristics of esophageal cortical evoked potentials (CEP) following electrical and mechanical stimulation in healthy subjects to evaluate the afferents involved in mediating esophageal sensation. Similarities in morphology and interpeak latencies of the CEP to electrical and mechanical stimulation suggest that they are mediated via similar pathways. Conduction velocity of CEP to either electrical or mechanical stimulation was 7.9-8.6 m/s, suggesting mediation via thinly myelinated Adelta-fibers. Amplitudes of CEP components to mechanical stimulation were significantly smaller than to electrical stimulation at the same levels of perception, implying that electrical stimulation activates a larger number of afferents. The latency delay of approximately 50 ms for each mechanical CEP component compared with the corresponding electrical CEP component is consistent with the time delay for the mechanical stimulus to distend the esophageal wall sufficiently to trigger the afferent volley. In conclusion, because the mechanical and electrical stimulation intensities needed to obtain esophageal CEP are similar and clearly perceived, it is likely that both spinal and vagal pathways mediate esophageal CEP. Esophageal CEP to both modalities of stimulation are mediated by myelinated Adelta-fibers and produce equally robust CEP responses. Both techniques may have important roles in the assessment of esophageal sensory processing in health and disease.     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.      

TGFβ3 and loading increases osteocyte survival in human cancellous bone cultured ex vivo

The goal of this study was to assess the effect of the addition of TGFβ₃, alone or in combination with loading, on the survival of osteocytes in 3D human explant cancellous bone during long-term culture in an ex vivo loading bioreactor. Human cancellous bone explants were cultured for up to 14 days with or without TGFβ₃ (15 ng ml⁻¹) and with or without loading (300 cycles, at 1 Hz, producing 4000 microstrain). Bone core response was visualized using undecalcified histology with morphological methods after embedding with Technovit 9100 New® resin. Histological examination revealed normal gross level bone structure with or without the application of load or the addition of TGFβ₃. The viability of the osteocytes within the bone was assessed by lactate dehydrogenase (LDH) activity. We demonstrate that this ex vivo loading bioreactor is able to maintain a high percentage (over 50%) of viable osteocytes throughout the bone explants after 14 days in ex vivo culture. Further to this, the combination of daily loading and TGFβ₃ administration produced superior osteocyte survival at the core centres when compared to loading or TGFβ alone. Copyright © 2008 John Wiley & Sons, Ltd.