Murine cathepsin D deficiency is associated with dysmyelination/myelin disruption and accumulation of cholesteryl esters in the brain

Cathepsin D (CTSD) deficiencies are fatal neurological diseases that in human infants and in sheep are characterized by extreme loss of neurons and myelin. To date, similar morphological evidence for myelin disruption in CTSD knockout mice has not been reported. Here, we show that CTSD deficiency leads to pronounced myelin changes in the murine brain: myelin-related proteolipid protein and myelin basic protein were both markedly reduced at postnatal day 24, and the amount of lipids characteristically high in myelin (e.g. plasmalogen-derived alkenyl chains and glycosphingolipid-derived 20- and 24-carbon acyl chains) were significantly lowered compared with controls. These changes were accompanied by ultrastructural alterations of myelin, including significant thinning of myelin sheaths. Furthermore, in CTSD knockout brains there was a pronounced accumulation of cholesteryl esters and abnormal levels of proteins related to cholesterol transport, with an increased content of apolipoprotein E and a reduced content of ATP-binding cassette transporter A1. These results provide evidence for dysmyelination and altered trafficking of cholesterol in brains of CTSD knockout mice, and warrant further studies on the role of lipid metabolism in the pathogenesis of CTSD deficiencies.     

PCR DGGE and RT-PCR DGGE show diversity and short-term temporal stability in the Clostridium coccoides-Eubacterium rectale group in the human intestinal microbiota

As the Clostridium coccoides-Eubacterium rectale (Erec; clostridial phylogenetic cluster XIVa) group is one of the major groups of the human intestinal microbiota, DNA- and RNA-based population analysis techniques (denaturing gradient gel electrophoresis; DGGE) were developed and applied to assess the diversity and temporal stability (6 months-2 years) of this faecal clostridial microbiota in 12 healthy adults. The stability of the Erec group was compared with the stability of the predominant bacterial microbiota, which was also assessed with PCR-DGGE. In addition, the Erec group was quantified with a hybridization-based method. According to our results, the Erec group was diverse in each subject, but interindividual uniqueness was not as clear as that of the predominant bacteria. The Erec group was found to be temporally as stable as the predominant bacteria. Over 200 clones obtained from two samples proved the developed method to be specific. However, the amount of bacteria belonging to the Erec group was not related to the diversity of that same bacterial group. In conclusion, the newly developed DGGE method proved to be a valuable and specific tool for the direct assessment of the stability of the Erec group, demonstrating diversity in addition to short-term stability in most of the subjects studied.     

Community Structure of the Bacteria Associated with Nodularia sp. (Cyanobacteria) Aggregates in the Baltic Sea

The community structure of the bacteria associated with Nodularia spumigena (Mertens) cyanobacterial aggregates in the Baltic Sea was studied with temperature gradient gel electrophoresis (TGGE), using a 16S rRNA gene fragment as a target. Various developmental stages of the aggregates and free-floating cyanobacterial filaments were sampled to reveal possible changes in associated microbial community structure during development and senescence of the aggregates. The microbial community structures of all samples differed, and the communities of young and decaying aggregates were separated by cluster analysis of the TGGE fingerprint data. Sequencing of the TGGE fragments indicated the presence of bacteria from the α-, β-, and γ-proteobacterial groups, as well as members of Cytophaga–Flexibacter–Bacteroides lineages and gram-positive Actinobacteria spp. The majority of the Nodularia-associated sequences were not closely related to previously reported 16S rDNA sequences from the Baltic Sea or any other environment. The structure of the bacterial assemblage reflects the environmental changes associated with the succession and decay of the cyanobacterial aggregates. In addition, the sequence data suggest that the N. spumigena (Mertens) blooms in the Baltic Sea may host thus far uncharacterized bacterial species.     

Interaction of huntingtin fragments with brain membranes--clues to early dysfunction in Huntington's disease

Abstract Huntingtin is a large, multi-domain protein of unknown function in the brain. An abnormally elongated polyglutamine stretch in its N-terminus causes Huntington's disease (HD), a progressive neurodegenerative disorder. Huntingtin has been proposed to play a functional role in membrane trafficking via proteins involved in endo- and exocytosis. Here, we supply evidence for a direct association between huntingtin and membranes. In the brains of R6/2 mice with HD pathology, a 64 kDa N-terminal huntingtin fragment accumulated in postsynaptic membranes during the pre-symptomatic period of 4-8 weeks of age. In addition, an oligomeric fragment of approximately 200 kDa was detected at 8 weeks of age. Simultaneous progressive changes in distribution of amphiphysin, synaptojanin, and subunits of NMDA- and AMPA-receptors provide a strong indication of dysfunctional synaptic trafficking. Composition of the major phospholipids in the synaptic membranes was unaffected. In vitro, large unilamellar vesicles of brain lipids readily associated with soluble N-terminal huntingtin exon 1 fragments and stimulated fibrillogenesis of mutant huntingtin aggregates. Moreover, interaction of both mutant and wild-type huntingtin exon 1 fragments with brain lipids caused bilayer perturbation, mediated through a proline-rich region adjacent to the polyglutamines. This suggests that lipid interactions in vivo could influence misfolding of huntingtin and may play an early role in HD pathogenesis.     

Succession of sea-ice bacterial communities in the Baltic Sea fast ice

Coastal fast ice and underlying water of the northern Baltic Sea were sampled throughout the entire ice winter from January to late March in 2002 to study the succession of bacterial biomass, secondary production and community structure. Temperature gradient gel electrophoresis (TGGE) and sequencing of TGGE fragments were applied in the community structure analysis. Chlorophyll-a and composition of autotrophic and heterotrophic assemblages were also examined. Overall succession of ice organism assemblages consisted of a low-productive stage, the main algal bloom, and a heterotrophic post-bloom situation, as typical for the study area. The most important groups of organisms in ice in terms of biomass were dinoflagellates, plasticidic flagellates, rotifers and ciliates. Ice bacteria showed a specific succession not directly dependent on the overall succession events of ice organisms. Sequenced 16S rDNA fragments were mainly affiliated to α-, β-, and γ-proteobacterial phyla and Cytophaga–Flavobacterium–Bacteroides-group, and related to sequences from cold environments, also from the Baltic Sea. Temporal clustering of the TGGE fingerprints was stronger than spatial, although lower ice and underlying water communities always clustered together, pointing to the importance of ice maturity and ice–water interactions in shaping the bacterial communities.     

Outer surface protein E antibody response and its effect on complement factor H binding to OspE in Lyme borreliosis

Borrelia burgdorferi sensu stricto and B. afzelii, but not B. garinii, are able to escape complement attack by binding factor H via OspE proteins. Recent finding of ospE genes also in B. garinii isolates has raised the question whether, under in vivo-conditions, B. garinii also expresses OspE proteins and consequently induces an antibody response. We set up an IgG ELISA by using recombinant OspE as an antigen. Sixty percent of acute and 64% of convalescent 25 erythema migrans patient samples were positive for anti-OspE antibodies. Anti-OspE antibodies were also found in the sera (83.6%) and cerebrospinal fluids (36%) of patients with neuroborreliosis. Since B. garinii is the major causative agent of neuroborreliosis, the result suggests that OspE is expressed by B. garinii in vivo. Of the 10 acrodermatitis chronica atrophicans patients, 80% had anti-OspE antibodies. Anti-OspE antibody positive sera inhibited factor H binding to Borrelia more efficiently than normal control sera (65% vs. 33.7%). Our results indicate that Borrelia spirochetes, including B. garinii, can induce the production of anti-OspE antibodies. This implies that OspE protein is produced in vivo by B. garinii possibly enabling it to escape complement and cause a CNS infection.     

Binding of complement regulators factor H and C4b binding protein to group A streptococcal strains isolated from tonsillar tissue and blood

Group A streptococcus (GAS) is the most common pathogen causing bacterial pharyngitis. We isolated streptococcal strains from tonsils removed from patients with tonsillar disease (n=202) and studied their ability to bind the complement regulators factor H (FH) and C4b binding protein (C4BP) using 125 I-labeled proteins. Blood isolates of GAS (n=10) were obtained from patients with bacteraemia. Streptococci were isolated from 21% of the tonsillitis patients. The emm and T types of the GAS strains were determined. Of the 26 GAS strains studied, only six could bind FH and/or C4BP above the threshold levels. The fraction of the offered radioactive protein bound ranged between 6-12% for FH and 19-56% for C4BP. The clinical course of the tonsillar disease was not related to the binding of FH or C4BP by GAS. The binding strains were mostly of the T4M4 or T28M28 type. From the invasive strains (n=10), three bound FH (binding level: 8-11%) and two C4BP (36-39%). The binding correlated only partially to M-protein (emm) type suggesting that the binding was not exclusively due to M-protein. The results indicate that complement regulator binding by GAS is only partially related to pathogenicity and not a universal property of all group A streptococci.