Environmental Factors Associated with Disease Progression after the First Demyelinating Event: Results from the Multi-Center SET Study

Objectives

To investigate the associations of environmental MS risk factors with clinical and MRI measures of progression in high-risk clinically isolated syndromes (CIS) after the first demyelinating event.

Methods

We analyzed 211 CIS patients (age: 28.9±7.8 years) enrolled in the SET study, a multi-center study of high-risk CIS patients. Pre-treatment samples were analyzed for IgG antibodies against cytomegalovirus (anti-CMV), Epstein Barr virus (EBV) early nuclear antigen-1 (EBNA-1), viral capsid antigen (VCA), early antigen-diffuse (EA-D), 25 hydroxy-vitamin D3 and cotinine levels and HLA DRB1*1501 status. The inclusion criteria required evaluation within 4 months of the initial demyelinating event, 2 or more brain MRI lesions and the presence of two or more oligoclonal bands in cerebrospinal fluid. All patients were treated with interferon-beta. Clinical and MRI assessments were obtained at baseline, 6, 12, and 24 months.

Results

The time to first relapse decreased and the number of relapses increased with anti-CMV IgG positivity. Smoking was associated with increased number and volume of contrast-enhancing lesions (CEL) during the 2-year period. The cumulative number of CEL and T2 lesions during the 2-year period was greater for individuals in the highest quartile of anti-EBV VCA IgG antibodies. The percent loss of brain volume was increased for those in the highest quartile of with anti-EBV VCA IgG antibodies.

Conclusions

Relapses in CIS patients were associated with CMV positivity whereas anti-EBV VCA positivity was associated with progression on MRI measures, including accumulation of CEL and T2 lesions and development of brain atrophy.     

Steroid-binding site of human and rabbit sex steroid binding protein of plasma: fluorescence characterization with equilenin

The interaction of the estrogen d-3-hydroxy-1,3,5(10),6,8-estrapentaen-17-one (equilenin) with the human and rabbit sex steroid binding proteins (hSBP and rSBP, respectively) has been investigated by using fluorescence and absorption spectroscopy. Equilenin competes for the binding of 5 alpha-dihydrotestosterone. The calculated binding constant of equilenin for rSBP is 1.9 X 10(7) M-1 at 4 degrees C, which can be compared with the binding constant of 5.7 X 10(7) M-1 reported for hSBP [Ross, J.B.A., Torres, R., & Petra, P.H. (1982) FEBS Lett. 149, 240]. The results of fluorescence quenching experiments with the collisional quenchers KI and acrylamide indicate that the bound steroid has limited accessibility to the bulk solvent and that there are no anionic surface groups near the steroid-binding site. The fluorescence excitation spectra of SBP-equilenin complexes are similar to the absorption spectra of equilenin in low-dielectric solvents. The fluorescence emission of the SBP-equilenin complexes, however, exhibits wavelength shifts (red shifts) opposite to those of the steroid in low-dielectric solvents or complexed with beta-cyclodextrin (blue shifts) but similar to the red shift produced by addition of the proton acceptor triethylamine to equilenin in cyclohexane. These data indicate that the steroid-binding site of hSBP and rSBP is a nonpolar cavity containing a proton acceptor that participates in a specific interaction, possibly a hydrogen bond, with the 3'-hydroxyl group of the bound steroid.     

Toxic and nontoxic microcystis colonies in natural populations can be differentiated on the basis of rRNA gene internal transcribed spacer diversity

Assessing and predicting bloom dynamics and toxin production by Microcystis requires analysis of toxic and nontoxic Microcystis genotypes in natural communities. We show that genetic differentiation of Microcystis colonies based on rRNA internal transcribed spacer (ITS) sequences provides an adequate basis for recognition of microcystin producers. Consequently, ecological studies of toxic and nontoxic cyanobacteria are now possible through studies of rRNA ITS genotypic diversity in isolated cultures or colonies and in natural communities. A total of 107 Microcystis colonies were isolated from 15 lakes in Europe and Morocco, the presence of microcystins in each colony was examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and they were grouped by rRNA ITS denaturing gradient gel electrophoresis (DGGE) typing. Based on DGGE analysis of amplified ITSa and ITSc fragments, yielding supplementary resolution (I. Janse et al., Appl. Environ. Microbiol. 69:6634-6643, 2003), the colonies could be differentiated into 59 classes. Microcystin-producing and non-microcystin-producing colonies ended up in different classes. Sequences from the rRNA ITS of representative strains were congruent with the classification based on DGGE and confirmed the recognition of microcystin producers on the basis of rRNA ITS. The rRNA ITS sequences also confirmed inconsistencies reported for Microcystis identification based on morphology. There was no indication for geographical restriction of strains, since identical sequences originated from geographically distant lakes. About 28% of the analyzed colonies gave rise to multiple bands in DGGE profiles, indicating either aggregation of different colonies, or the occurrence of sequence differences between multiple operons. Cyanobacterial community profiles from two Dutch lakes from which colonies had been isolated showed different relative abundances of genotypes between bloom stages and between the water column and surface scum. Although not all bands in the community profiles could be matched with isolated colonies, the profiles suggest a dominance of nontoxic colonies, mainly later in the season and in scums.     

Spectroscopic study on the communication between a heme a3 propionate, Asp399 and the binuclear center of cytochrome c oxidase from Paracoccus denitrificans

The proton pumping mechanism of cytochrome c oxidase on a molecular level is highly disputed. Recently theoretical calculations and real time electron transfer measurements indicated the involvement of residues in the vicinity of the ring A propionate of heme a3, including Asp399 and the CuB ligands His 325, 326. In this study we probed the interaction of Asp399 with the binuclear center and characterize the protonation state of its side chain. Redox induced FTIR difference spectra of mutations at the site in direct comparison to wild type, indicate that below pH 5 Asp 399 displays signals typical for the deprotonation of the acidic residue with reduction of the enzyme. Interestingly at a pH higher than 5, no contributions from Asp 399 are evident. In order to probe the interaction of the site with the binuclear center we followed the rebinding of CO by infrared spectroscopy for mutations on residue Asp399 to Glu, Asn and Leu. Previously different CO conformers have been identified for bacterial cytochrome c oxidases, and its pH dependent behaviour discussed to be relevant for catalysis. Interestingly we observe the lack of this pH dependency and a strong influence on the observable conformers for all mutants studied here, clearly suggesting a communication of the site with the heme-copper center and the nearby histidine residues.     

Spatial analyses of two color polymorphisms in an alpine grasshopper reveal a role of small‐scale heterogeneity

Discrete color polymorphisms represent a fascinating aspect of intraspecific diversity. Color morph ratios often vary clinally, but in some cases, there are no marked clines and mixes of different morphs occur at appreciable frequencies in most populations. This poses the questions of how polymorphisms are maintained. We here study the spatial and temporal distribution of a very conspicuous color polymorphism in the club‐legged grasshopper Gomphocerus sibiricus. The species occurs in a green and a nongreen (predominately brown) morph, a green–brown polymorphism that is common among Orthopteran insects. We sampled color morph ratios at 42 sites across the alpine range of the species and related color morph ratios to local habitat parameters and climatic conditions. Green morphs occurred in both sexes, and their morph ratios were highly correlated among sites, suggesting shared control of the polymorphism in females and males. We found that in at least 40 of 42 sites green and brown morphs co‐occurred with proportions of green ranging from 0% to 70% with significant spatial heterogeneity. The proportion of green individuals tended to increase with decreasing summer and winter precipitations. Nongreen individuals can be further distinguished into brown and pied individuals, and again, this polymorphism is shared with other grasshopper species. We found pied individuals at all sites with proportions ranging from 3% to 75%, with slight, but significant variation between years. Pied morphs show a clinal increase in frequency from east to west and decreased with altitude and lower temperatures and were more common on grazed sites. The results suggest that both small‐scale and large‐scale spatial heterogeneity affects color morph ratios. The almost universal co‐occurrence of all three color morphs argues against strong effects of genetic drift. Instead, the data suggest that small‐scale migration–selection balance and/or local balancing selection maintain populations polymorphic.     

Involvement of fibronectin during epiboly and gastrulation in embryos of the common carp,Cyprinus carpio

Summary The present report firstly describes a pilot study in which, during early development of embryos of the common carp, Cyprinus carpio, the cellular adhesion to fibronectin (FN) was blocked by administration of GRGDS peptide (which binds to the FN-receptor). As this treatment resulted in developmental aberrations, suggesting a functional role for FN, the major part of the work was focussed on the distribution of reactivity of anti-FN antibodies during epiboly and gastrulation. GRGDS treatment had a concentration dependent effect on development. Incubation of embryos in 1.5 mg/ml from the 32-cell stage onwards caused a retardation of epiboly, which did not proceed beyond 60%. The embryos did not show involution, as was confirmed by histological study. These preliminary results suggest that FN is involved in both epiboly and gastrulation of carp embryos. During cleavage, no specific extracellular binding of anti-FN antiserum could be observed. However, binding to a number of cell membranes took place from early epiboly onwards. After the onset of gastrulation, we observed a gradually increasing number of the deepest epiblast cells, showing immunostaining on part of their surface, facing the yolk syncytial layer (YSL) or the involuted cells. During early epiboly, anti-FN binding was restricted to areas in front of the migratory hypoblast cells. Later on, binding was found at the border of hypoblast and epiblast cells. At 100% epiboly, some contact areas of epiblast and hypoblast showed a discontinuous lining of reactivity, whilst other areas appeared devoid of anti-FN binding sites. The results indicate that FN is involved in the migration and guidance of hypoblast cells during gastrulation in carp. Correspondence to: P. Gevers